Mobility-modified nucleobase polymers and methods of using same

ABSTRACT

The present invention relates generally to nucleobase polymer functionalizing reagents, to mobility-modified sequence-specific nucleobase polymers, to compositions comprising a plurality of mobility-modified sequence-specific nucleobase polymers, and to the use of such polymers and compositions in a variety of assays, such as, for example, for the detection of a plurality of selected nucleotide sequences within one or more target nucleic acids. The mobility-modifying polymers of the present invention include phosphoramidite reagents which can be joined to other mobility-modifying monomers and to sequence-specific oligonucleobase polymers via uncharged phosphate triester linkages. Addition of the mobility-modifying phosphoramidite reagents of the present invention to oligonucleobase polymers results in unexpectedly large effects the mobility of those modified oligonucleobase polymers, especially upon capillary electrophoresis in non-sieving media.

1. FIELD OF THE INVENTION

The present invention relates generally to nucleobase polymerfunctionalizing reagents, to mobility-modified sequence-specificnucleobase polymers, to compositions comprising a plurality ofmobility-modified sequence-specific nucleobase polymers, and to the useof such polymers and compositions in a variety of assays, such as, forexample, for the detection of a plurality of selected nucleotidesequences within one or more target nucleic acids.

2. BACKGROUND OF THE INVENTION

Methods used to detect selected nucleotide sequences within targetnucleic acids underlie an extensive array of practical applicationsincluding, but not limited to, paternity testing, forensic analysis,organ donor recipient matching, disease diagnosis, prognosis andtreatment, and prenatal counseling.

There exists a need in the art for materials and methods that permitpluralities of selected nucleotide sequences to be simultaneouslydetected and analyzed, under uniform experimental conditions, preferablyin a single, automated, assay reaction. One approach towards meetingthis need has been the development of mobility-modifying polymers thatcan be attached to sequence-specific nucleobase polymers that act toincrease the effective size of the modified nucleobase polymers. Wherethe charge to translational frictional drag ratio of themobility-modifying polymer differs from that of the nucleobase polymerto which it is attached, the resulting modified nucleobase polymer willhave an electrophoretic mobility that differs from that of theunmodified nucleobase polymer. This alteration of the charge totranslational frictional drag ratio may be employed in variousapplications to effect electrophoretic separation of similarly-sizednucleobase polymers under both sieving and non-sieving conditions.

The most commonly employed mobility-modifying polymers are polyethyleneoxides (PEO) that are attached to a nucleobase polymer using standardDNA chemistry (Grossman et al. (1994) Nucleic Acids Research 22 (21):4527-34). An exemplary standard PEO phosphoramidite reagent (“PEOreagent”) that can be added to a nucleobase polymer using standard DNAchemistry is illustrated below:

In the illustration, DMT represents dimethoxytrityl and iPr representsisopropyl. When x=5, each PEO reagent added to the nucleobase polymerimparts the nucleobase polymer with an electrophoretic retardation ofapproximately 2 nucleotides as compared to the unmodified nucleobasepolymer under both sieving and nonsieving electrophoretic conditions.Due to limitations of DNA chemistry, no more than about 40 PEO reagentscan be coupled to a nucleobase polymer and result in homogenous product.Accordingly, the greatest electrophoretic mobility retardation that canbe achieved using these standard PEO modifying reagents is about 80nucleotides.

However, in light of the increasing need to simultaneously analyze vastnumbers of nucleotide sequences in a single experiment, e.g., the 200identified alleles associated with cystic fibrosis, there remains a needin the art for new mobility-modifying polymers that have differentcharge to translational frictional drag ratios than currently availablemobility-modifying polymers, and that can impart electrophoreticmobility retardations of greater than the 80 nucleotides achievable withavailable PEO modifying reagents. The availability of such newmobility-modifying polymers would greatly increase the repertoire ofavailable mobility modifications, thereby enabling the ability toperform extremely complex sequence analyses in simple, preferablyautomated, formats.

3. SUMMARY OF THE INVENTION

In one aspect, the present invention provides mobility-modifyingphosphoramidite functionalizing reagents comprising a polymeric portionand a phosphoramidite moiety. The phosphoramidite moiety comprises anoxygen-protecting group that, quite unlike the β-cyanoethyl oxygenprotecting group used in conventional phosphoramidite reagents, isstable to basic conditions such as the conditions and reagents used inconventional phosphoramidite oligonucleotide synthesis and deprotection.As a consequence of this stable oxygen protecting group, the bond formedbetween the functionalizing reagent and the compound functionalized isan uncharged phosphate triester. The mobility-modifying phosphoramiditereagents of the invention may be used to functionalize a wide variety ofsubstances and materials to add mass and size to the substance ormaterial without substantially altering its overall net charge.

The mobility-modifying phosphoramidite reagents of the invention arecompatible with standard phosphoramidite synthetic schemes and can beused with commercially available nucleobase polymer synthesisinstruments. Thus, the mobility-modifying phosphoramidite reagents ofthe present invention are particularly convenient for mobility-modifyingsynthetic sequence-specific nucleobase polymers such as, for example,2′-deoxyoligonucleotides. They may be readily attached to the5′-terminus of the nucleobase polymer, to the 3′-terminus, or to boththe 5′- and 3′-termini, depending on the particular application and/ordesired degree of mobility modification.

The terminus of the polymeric portion that is distal to thephosphoramidite moiety may be protected with a group that is selectivelyremovable under the desired synthesis conditions, or it may comprise agroup that is essientially non-reactive, such as, for example an alkyl,aryl, arylakyl, etc. group. In the former embodiment, the protectinggroup may be selectively removed for sequential condensation of one ormore additional phosphoramidite reagents. Suitable selectively removableprotecting groups will depend upon the identity of the group beingprotected and will be apparent to those of skill in the art. Selectivelyremovable groups suitable for protecting hydroxyl groups include, by wayof example and not limitation, any of the acid-labile groups that arecommonly used to protect the 5′-hydroxyl of conventional nucleosidephosphoramidites oligonucleotide synthesis reagents, such as acid-labiletrityl groups (e.g., monomethoxytrityl, dimethoxytrityl, etc.).

The mobility-modifying phosphoramidite reagents of the invention may beused alone to mobility-modify substances such as nucleobase polymers toadd mass and size to the nucleobase polymer without altering its overallnet charge. Alternatively, they may be used in conjunction withconventional mobility-modifying reagents, such as the PEO reagentillustrated above, to mobility-modify substances such as nucleobasepolymers. Because the mobility-modifying phosphoramidite reagents of theinvention may be used to add mass and size to a substance such as anucleobase polymer without altering its overall charge, when used inconjunction with conventional reagents such as PEO reagents, they vastlyincrease the repertoire of available mobility-modifications that can beadded to substances such as nucleobase polymers.

The polymeric portion composing the reagent may be any of a variety ofpolymers that are soluble under the desired conditions of use and thateither include, or can be modified to include, a functional group, suchas, for example, a primary hydroxyl group, that can be convenientlyconverted to a phosphoramidite moiety, typically using standardart-known chemistries. Typical polymers include, but are not limited to,polyoxides, polyamides, polyimines and polysaccharides. The polymers maybe used singly or in combinations, such as in the form of copolymers orblock polymers. Exemplary polymers include linear or branchedpolyalkylene oxides, or derivatives thereof, comprising from about 2 to10 monomer units. Typical derivatives include, for example, those inwhich the terminal hydroxyl is replaced with a sulfanyl group or anamino group. A useful polyalkylene oxide polymer is polyethylene glycol.The polymer may optionally include a label or other reporter group ormolecule, a protecting group for protecting the mobility-modifiednucleobase polymer during subsequent synthesis reactions, or othergroups for, e.g., binding the mobility-modified nucleobase polymer toother moieties or chemical species, such as, e.g., ligands, etc.

As discussed above, the group protecting the phosphoramidite oxygen atomof the reagents of the invention is stable to the basic conditions usedto deprotect and/or cleave synthetic nucleobase polymers such asoligonucleotides. Thus, the oxygen protecting group should generally bestable to treatment with ammonium hydroxide at a temperature of 55° C.for a period of about 18 hrs. Of course, if milder deprotection and/orcleavage conditions are used, the oxygen protecting group need only bestable to these milder conditions. Groups stable to such basicconditions that can be used to protect the oxygen atom of thephophoramidite reagents of the invention will be apparent to those ofskill in the art, and include by way of example and not limitation,alkyls comprising at least two carbon atoms, aryls and (R⁸)₃Si— whereeach R⁸ is independently selected from the group consisting of linearand branched chain alkyl and aryl. Alternatively, the oxygen protectinggroup may be a polymer segment, optionally having a selectivelyremovable terminal protecting group as described above. In this latterembodiment, the reagents of the invention permit the formation ofmobility-modified substances comprising branched or dendritic polymersegments.

In one convenient embodiment, the polymer portion of themobility-modifying phosphoramidite reagents of the present invention isa polyalkylene oxide, an illustrative embodiment of which is depicted asFormula (I) below:

wherein:

R⁵ is selected from the group consisting of hydrogen, protecting group,reporter molecule, and ligand;

X is selected from the group consisting of O, S, NH, and NH—C(O);

each a is, independently, an integer from 1 to 6;

b is an integer from 0 to 40;

R⁶ and R⁷ are each independently selected from the group consisting ofC₁-C₆ alkyl, C₃-C₁₀ cycloalkyl, C₆-C₂₀ aryl, and C₂₀-C₂₇ arylalkyl; and

R² is selected from the group consisting of alkyl comprising at leasttwo carbon atoms, aryl, (R⁸)₃Si— where each R⁸ is independently selectedfrom the group consisting of linear and branched chain alkyl and aryl,base-stable protecting groups, and R⁵—X—[(CH₂)_(a)—O]_(b)—(CH₂)_(a)—.

In another aspect, the present invention provides sequence-specificnucleobase polymers that have been mobility-modified with themobility-modifying phosphoramidite reagents of the invention, eitheralone or in combination with conventional mobility-modified reagents.Such sequence-specific mobility-modified nucleobase polymers generallycomprise a mobility-modifying polymeric segment and a sequence-specificnucleobase segment.

The nucleobase polymer segment is typically an oligonucleotide such as aDNA oligomer or an RNA oligomer, but may also be any of a number ofdifferent analogs or derivatives of DNA and/or RNA, as will be describedin more detail in a later section. The nucleobase polymer has a sequenceof nucleobases that is at least partially complementary to a desirednucleotide sequence of a target nucleic acid such that the nucleobasepolymer segment specifically binds the target sequence, under specifiedconditions.

The mobility-modifying polymer segment has a ratio ofcharge-to-translational frictional drag that is different from that ofthe nucleobase polymer segment in a given electrophoretic medium.Consequently by virtue of the mobility-modifying polymer segment, themobility-modified sequence-specific nucleobase polymers of the inventionhave electrophoretic mobilities that are retarded as compared with thoseof the corresponding unmodified nucleobase polymer.

According to one illustrative embodiment of the invention, themobility-modified sequence-specific nucleobase polymer is a compoundaccording to structural formula (II):

or a salt thereof, wherein:

R², R⁵, X, a, and b are as in Formula (I); and

OLIGO is a sequence-specific nucleobase polymer comprising at least fivenucleobases.

The mobility-modifying polymer can be attached to the 5′-end, the3′-end, or both the 5′-end and the 3′-end of the OLIGO. Themobility-modifying polymer can also be attached to the 5′-end of a firstnucleobase polymer and to the 3′-end of a second nucleobase polymer,thereby providing a mobility-modified oligonucleobase polymer having themobility-modifying polymer segment linking two nucleobase polymersegments.

By virtue of substituent R² in the mobility-modified nucleobase polymersaccording to Formula (II), the illustrated phosphate triester isuncharged at physiological pH. The illustrated mobility-modifyingpolymeric group, therefore, would add only mass, and not charge, to theOLIGO.

The mobility-modified nucleobase polymer of the invention are notlimited to those including only uncharged phosphate triester linkages.By judiciously selecting combinations of uncharged phosphate triesterlinkages and charged phosphate diester linkages, the repertoire ofavailable mobility-modifications can be dramatically increased.Accordingly, in a second illustrative embodiment, the mobility-modifiedsequence-specific nucleobase polymer is a compound according tostructural formula (III):

or a salt thereof, wherein:

R², R⁵, a, b, X, and OLIGO are as previously defined in Formulae (I) and(II);

each d is independently an integer from 1 to 200; and

each R⁴ and each R¹⁰ is independently selected from the group consistingof hydrogen and R², with the proviso that at least one R¹⁰ or at leastone R⁴ is other than hydrogen.

In the compounds of structural formula (III), each a, b, d, X, R¹⁰ andR⁴ may, independently of one another, be the same or different. Incertain embodiments of the compounds of structural formulae (III), atleast some R¹⁰ and/or R⁴ are other than hydrogen. Where R¹⁰ and R⁴ areother than hydrogen, R¹⁰ and/or R⁴ are R².

In another embodiment, the present invention provides compositionscomprising a plurality of mobility-modified sequence-specific nucleobasepolymers of the invention, wherein, in certain embodiments, each saidmobility-modified nucleobase polymer has a structure independentlyselected from the group consisting of structural formulae (II) and(III). At least two of the mobility-modified nucleobase polymers of theplurality has a distinctive ratio of charge to translational frictionaldrag such that each of the two or more mobility-modified nucleobasepolymers has a distinct electrophoretic mobility. The distinctive ratiosof charge to translational frictional drag may be due to differences inthe lengths (i.e., the number of monomer units) of themobility-modifying polymer segment of the molecule, differences in thenumber of mobility-modifying polymer segments attached to the nucleobasepolymer (i.e., differences in: variable d in structural formulae (II)and (III)), the charges linking multiple mobility-modifying polymersegments, the number of charged versus uncharged subunits, the lengthand charge of the nucleobase polymer, or a combination of thesefeatures.

In yet another aspect, the present invention provides a method ofdetecting a plurality of nucleotide sequences within one or more targetnucleic acids. According to the method, a plurality of mobility-modifiedsequence-specific nucleobase polymer probes, each of which optionallyhas a structure independently selected from the group consisting ofstructural formula (II) and (III), is contacted with one or more targetnucleic acids, generally under conditions that distinguish thosemobility-modified probes that hybridize to the target nucleic acid in abase-specific manner from those that do not. The mobility-modifiednucleobase polymer probes that hybridize to the target are thenfractionated by electrophoresis. The presence of selected sequence(s) inthe target nucleic acid is detected according to the observedelectrophoretic migration rates of the mobility-modified nucleobasepolymer probes, or, optionally, according to the identity of a label orby a combination thereof.

The mobility-modified nucleobase polymer probes may be either labeled orunlabeled. Alternatively, they may be modified to include a label duringthe method, as well be described more fully below. When unlabeled, theelectrophoretic migration rates of the mobility-modified probes may bemonitored by conventional means, for example by absorbance spectroscopy.When labeled, for example with a fluorophore, the electrophoreticmigration rates may be monitored by detecting the label.

In one embodiment of the present invention, in order to facilitatedetection of the mobility-modified probes in a multiplex assay, themobility-modified probes are labeled are labeled with differentfluorescent labels such as, but not limited to, 5-carboxyfluorescein(5-FAM), 6-carboxy-fluorescein (6-FAM),2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE),N,N,N′-N-tetramethyl-6-carboxy rhodamine (TAMRA), 6-carboxy-X-rhodamine(ROX), 4,7,2′,4′,5′,7′-hexachloro-6-carboxy-fluorescein (HEX-1),4,7,2′,4′,5′,7′-hexachloro-5-carboxy-fluorescein (HEX-2),2′,4′,5′,7′-tetrachloro-5-carboxy-fluorescein (ZOE),4,7,2′,7′-tetrachloro-6-carboxy-fluorescein (TET-1),1′,2′,7′,8′-dibenzo-4,7-dichloro-5-carboxyfluorescein (NAN-2), and1′,2′,7′,8′-dibenzo-4,7-dichloro-6-carboxyfluorescein. Guidance forselecting appropriate fluorescent labels can be found in Smith et al.(1987) Meth. Enzymol. 155:260-301, Karger et al. (1991) Nucl. Acids Res.19:4955-4962, Haugland (1989) Handbook of Fluorescent Probes andResearch Chemicals (Molecular Probes, Inc., Eugene, Oreg.). Exemplaryfluorescent labels include fluorescein and derivatives thereof, such asthose disclosed in U.S. Pat. No. 4,318,846 to Khanna et al. and Lee etal. (1989) Cytometry 10:151-164, and 6-FAM, JOE, TAMA, ROX, HEX-1,HEX-2, ZOE, TET-1 or NAN-2, as described above, and the like. When aplurality of fluorescent dyes are employed, they should, in many casesbe spectrally resolvable.

In one convenient embodiment of the method, the target nucleic acid(s)are immobilized on a solid support. Following hybridization,unhybridized probes are removed, typically by washing, and thehybridized probes are recovered, typically by denaturing the hybrids,and those recovered hybridized probes are fractionated byelectrophoresis as described above.

In another embodiment of the method, mobility-modified probes thatspecifically hybridize to the target nucleic acid are modified toincorporate a label. The modification may be accomplished in numerousdifferent ways, see, for example, U.S. Pat. Nos. 5,807,682, 5,703,222,and 5,470,705, which disclose methods and compositions useful for theselective modification of probes when bound to a target nucleic acid ina base-specific manner. In one general method a second sequence-specificnucleobase polymer probe which includes a detectable label is covalentlyjoined to the hybridized mobility-modified probe. This second labeledprobe may also optionally include a mobility-modifying polymer. The twoprobes can be covalently joined to one another when they both adjacentlyhybridize to the same target nucleic acid molecule (i.e., the probeshave confronting terminal nucleobase residues that basepair withadjacent bases of the target nucleic acid). The covalent joining may beaccomplished by chemical means or biological means, such as by a DNA orRNA ligase.

Thus, according to this aspect of the invention, the target nucleicacid(s), which may be in solution or immobilized, are contacted with afirst plurality of mobility-modified sequence-specific nucleobasepolymer probes according to the invention, each of which has adistinctive ratio of charge to translational frictional drag and is,optionally, selected from the group consisting of structural formula(II) and (III), and a second, labeled sequence-specific nucleobasepolymer probe, generally under conditions that distinguish those probesthat hybridize to the target in a sequence-specific manner. Probes thatadjacently hybridize to the same target nucleic acid molecule are thencovalently joined together (ligated) to form a mobility-modified labeledligation product. Each labeled ligation product has a distinctive ratioof charge to translational frictional drag. In a further aspect, threeor more nucleobase polymer probes are hybridized to adjacent sequencesof a target nucleic acid in such a manner that at least three probes canbe covalently joined to form a ligation product, wherein at least one ofthe probes so joined comprises a detectable label, and at least one ofthe probes so joined is a mobility-modified sequence-specific nucleobasepolymer probe, optionally selected from the group consisting ofstructural formula (II) and (III) such that the ligation product bears alabel and has a distinctive ratio of charge translational frictionaldrag. The labeled ligation products, which are hybridized to the targetnucleic acid, are recovered and fractioned by electrophoresis, asdescribed above.

This cycle of hybridization, joining, and denaturation, may be repeatedin order to amplify the concentration of the ligation product formed. Inthis instance, the joining may be accomplished by means of athermostable ligase. Furthermore, additional nucleobase polymer probes,which together are sufficiently complementary to the ligated product tohybridize thereto and be covalently joined to one another as above, arealso included, thereby affording geometric amplification of the ligatedproduct, i. e., a ligase chain reaction. The product of such a ligasechain reaction therefore is a double stranded molecule consisting of twostrands, each of which is the product of the joining of at least twosequence-specific nucleobase polymer probes. Accordingly, in yet anotheraspect of the present invention, at least one of the sequence-specificnucleobase polymers incorporated within the ligase chain reactionproduct comprises a detectable label, and at one of thesequence-specific nucleobase polymers is a mobility-modifiedsequence-specific nucleobase polymer selected from the group consistingof structural formula (II) and (III) such that at least one strand ofthe ligase chain reaction product has a distinctive ratio of charge totranslational frictional drag.

In a second general method, the modification is achieved via atemplate-directed fill-in reaction or via PCR. In this aspect, a targetnucleic acid, which may be in solution or immobilized, is contacted witha plurality of sequence-specific nucleobase polymers probes, two ofwhich hybridize to opposite ends of complementary strands flanking anucleotide sequence of interest within the target nucleic acid. Repeatedcycles of extension of the hybridized sequence-specific probes,optionally by a thermostable polymerase, thermal denaturation anddissociation of the extended product, and annealing, provide a geometricamplification of the region bracketed by the two nucleobase polymerprobes. The product of such a polymerase chain reaction therefore is adouble stranded molecule consisting of two strands, each of whichcomprises a sequence-specific nucleobase polymer probe. In this aspectof the present invention, at least one of the sequence-specificnucleobase polymer probes is a mobility-modified sequence-specificnucleobase polymer probe according to the invention, optionally a probeselected from the group consisting of structural formula (II) and (III),such that the double stranded polymerase chain reaction product has adistinctive ratio of charge to translational frictional drag. Thepolymerase chain reaction product formed in this aspect of the inventionmay further comprise a label, which may be incorporated within either ofthe sequence-specific nucleobase polymer probes used as primers, or itmay be incorporated within the substrate deoxyribonucleosidetriphosphates used by the polymerizing enzyme. In other instances, thepolymerase chain reaction product may be labeled by intercalation withan intercalating dye or by other non-covalent association with adetectable indicator molecule. In yet another aspect, the polymerasechain reaction product formed is analyzed under denaturing conditions,providing separated single stranded products. In this aspect, at leastone of the single stranded products comprises both a label and amobility-modified sequence-specific nucleobase polymer of the invention,optionally selected from the group consisting of structural formula (II)and (III) such that the single stranded product derived from doublestranded polymerase chain reaction product has a distinctive ratio ofcharge to translational frictional drag. As is well known in the art,such a single stranded product may also be generated by carrying out thePCR reaction with limiting amounts of one of the two sequence-specificnucleobase polymer probes used as a primer.

In a third general embodiment, bound mobility-modified probes arereacted with reporter-labeled nucleotide triphosphate molecules, in thepresence of a DNA polymerase to attach reporter groups, which includebut are not limited to radioactive and fluorescent moieties, to the 3′end of the probes.

In a fourth general embodiment, each mobility-modified probe includes asequence that may be enzymatically cleaved when the probe is bound to atarget nucleic acid. The cleavage reaction may remove a portion of thenucleobase polymer segment to modify the probe's ratio ofcharge/translational frictional drag, or may separate a reporter labelcarried at one end of the probe from a polymer chain carried at theother end of the probe to modify the charge/translational frictionaldrag of the portion carrying the reporter label. One method fordetecting such events relies upon a process, referred to as fluorescenceenergy resonance transfer (FRET), in which energy is passed between afluorophore donor and an acceptor molecule. Therefore, in one aspect ofthis embodiment, the mobility-modified probe comprises two moieties,separated by the cleavage site, which serve as photon donor andacceptor. Where the acceptor molecule is not a fluorophore, the effectis the quenching of donor fluorescence. Cleavage of the boundmobility-modified probe bound to the target nucleic acid physicallyseparates the donor and acceptor moieties and restores fluorescence bythe donor moiety, which is readily, and sensitively detected.

In still another aspect of the fourth general embodiment, eachmobility-modified probe, which includes a sequence that may be cleavedwhen the probe is bound to a target nucleic acid, comprises a firstmobility-modifying polymer attached to the labeled terminus of theprobe, which can be either the 5′-end or the 3′-end of the probe, and asecond mobility-modifying polymer attached to the unlabeled terminus ofthe probe. This aspect of the present invention is illustrated, in anon-limiting manner, by the use of the mobility-modifying polymers ofthe present invention in “invader assays,” which are SNP-identifyingprocedures based upon flap endonuclease cleavage of structures formed bytwo overlapping nucleobase polymers that hybridize to a target nucleicacid (see e.g. Cooksey et al., 2000, Antimicrobial Agents andChemotherapy 44: 1296-1301). Such cleavage reactions release productscorresponding to the 5′-terminal nucleobase(s) of the “downstream”nucleobase polymer. Where those cleavage products are labeled and can beseparated from the uncleaved nucleobase polymer, an invader assay can beused to discriminate single base differences in, for example, genomicsequences or PCR-amplified genomic sequences.

Attachment of the mobility-modifying polymers of the present inventionto the labeled 5′-terminus of the downstream nucleobase polymer used inan invader assay provides cleavage products with distinctive charge totranslational frictional drag ratios. Accordingly, a plurality of SNP'sare analyzed simultaneously using a plurality of sequence-specificdownstream nucleobase polymers, wherein the sequence-specific downstreamnucleobase polymers comprise a mobility-modifying polymer of the presentinvention attached to the labeled 5′-terminus, such that the productgenerated by flap endonuclease cleavage at each SNP has a distinctivecharge to translational frictional drag ratio.

In a further aspect of the invader assay, for example, the downstreamnucleobase polymer, which carries a label and a first mobility-modifyingpolymer of the present invention attached to the 5′-terminus, furthercomprises a second mobility-modifying polymer attached to the3′-terminus. The presence of the second mobility-modifying polymerincreases the sensitivity of the invader assay by enhancing thedifference between the electrophoretic mobility of the flap endonucleasegenerated product, comprising the 5′-terminus, label, and firstmobility-modifying polymer, and the electrophoretic mobility of theuncleaved downstream nucleobase polymer. Accordingly, the secondmobility-modifying polymer has a molecular weight of at least 2000. Inother embodiments, the second mobility-modifying polymer has a molecularweight of at least 5,000, at least 10,000, at least 20,000, and at least100,000. In one embodiment, the second mobility-modifying polymer is amobility-modifying polymer of the present invention, while in otherembodiments, the second mobility-modifying polymer is amobility-modifying polymer of the art, which is, in one illustrative,non-limiting example, an uncharged mono methyl polyethyleneglycolpolymer. Moreover, the second mobility-modifying polymer may comprise amixture of species of different molecular weight, provided that thosespecies do not interfere substantially with detection of the signalproduct, i.e., the flap endonuclease generated product, comprising the5′-terminus, label, and first mobility-modifying polymer (see Example 5,below).

In a fifth general embodiment, bound mobility-modified probes arecontacted with reporter molecules, including but not limited tointercalating dyes, that bind in a non-covalent manner to the duplex DNAstructure formed between the probe and target nucleic acids. Suchreporter molecules may form fluorescent complexes when bound to duplexDNA structures, or the non-covalently bound reporter molecule maycomprise, for example, a radioactive moiety or other detectable moiety,or a chemical group forming one member of a cognate binding pair,thereby modifying those mobility-modified probes that have bound to atarget nucleic acid.

In yet another aspect, the present invention provides a method forseparating target nucleic acid molecules, which may comprise differentnumbers of nucleotide residues, but nevertheless have substantially thesame ratio of charge to translational frictional drag. This methodcomprises contacting a mixture of target nucleic acid molecules with amobility-modified sequence-specific nucleobase polymer, optionallyhaving a structure selected from the group consisting of structuralformula (II) and (III); attaching the mobility-modifiedsequence-specific nucleobase polymer to substantially all the targetnucleic acids forming mobility-modified target nucleic acids, therebyproviding each target nucleic acid molecule having the same number ofnucleotide residues with a distinctive ratio of charge to translationfrictional drag; and separating the mobility-modified target molecules.Generally, the mobility-modified target molecules so formed areseparated by electrophoresis, e.g. by capillary electrophoresis, or bycapillary electrophoresis in a non-sieving medium.

A mixture of such target molecules is generated, in one embodiment, bychain termination or chemical cleavage sequencing reactions, in whichcase the target nucleic acids generally comprise at least one detectablelabel. Where the target molecules are generated by chain terminationreactions, those target molecules are primer extension products. In thisaspect, the primer extension products and the sequence-specificmobility-modified nucleobase polymer are further contacted undersuitable conditions with a template nucleic acid. The template nucleicacid has a 3′-region comprising at least 4 nucleotide residues and a5′-region comprising at least 4 nucleotide residues, wherein the3′-region is complementary to 3′-terminal residues of themobility-modified sequence-specific nucleobase polymer, and wherein the5′-region is complementary to 5′-terminal residues of each of the primerextension products, such that the 3′-terminal residue of themobility-modified sequence-specific nucleobase polymer abuts the5′-terminal residue of a primer extension product, when themobility-modified nucleobase polymer and the primer extension productare hybridized to the template nucleic acid. Therefore, in thisembodiment, the template nucleic acid is used to align themobility-modified sequence-specific nucleobase polymer and primerextension products under appropriate conditions, so that the hybridized,aligned nucleobase polymers can be covalently joined to one another,optionally by enzymatic ligation. The product formed thereby comprises amobility-modified sequence-specific nucleobase polymer and a primerextension product, providing a mobility-modified primer extensionproduct having a distinctive ratio of charge to translational frictionaldrag. Generally, in this aspect of the invention the primer extensionproduct will further comprise at least one detectable label. Thedetectable label, which may be incorporated into one or more of themobility-modified sequence-specific primer, deoxyribonucleotidesubstrate(s) or dideoxyribonucleotide substrate(s), may be, asnon-limiting examples, a radioactive label or a fluorescent label. Inone embodiment, the chain termination sequencing reaction comprises eachof the four dideoxyribonucleotide substrates, wherein each is labeledwith a different fluorescent moiety and wherein each of the fourdifferent fluorescent moieties are spectrally resolvable from oneanother. In other aspects of this embodiment, four separate chaintermination sequencing reactions are carried out, wherein each of thosefour reactions comprises a single dideoxyribonucleotide substrate and amobility-modified sequence specific primer carrying a single,spectrally-resolvable fluorescent moiety. In this instance, thesequencing reactions are terminated and combined to provide a mixture ofprimer extension products each of which is terminated with adideoxyribonucleotide residue and is labeled with aspectrally-resolvable fluorescent moiety that corresponds to thatdideoxyribonucleotide residue.

In another aspect of this method, the template nucleic acid employed hasa 3′-region comprising at least 4 nucleotide residues, a 5′-regioncomprising at least 4 nucleotide residues, and a central region disposedbetween the 3′-region and the 5′-region, wherein the 3′-region of thetemplate nucleic acid is complementary to 3′-terminal residues of themobility-modified sequence-specific nucleobase polymer, and wherein the5′-region of the template nucleic acid is complementary to 5′-terminalresidues of each of the primer extension products. In this instance,hybridization of the sequence-specific mobility-modified nucleobasepolymer and a primer extension product to the template nucleic acidmolecule, provides a structure in which the 3′-terminal residue of themobility-modified sequence-specific nucleobase polymer is separated fromthe 5′-terminal nucleotide residue of a primer extension product by anucleobase sequence corresponding to the central region of the templatenucleic acid. In this instance, a gap remains between the hybridizednucleobase polymers which is filled with a DNA polymerase in thepresence of at least one deoxyribonucleoside triphosphate substrate,thereby forming an extended mobility-modified sequence-specificnucleobase polymer. Upon filling of this gap, the extendedmobility-modified sequence-specific nucleobase polymer is ligated to the5′-terminal nucleotide residue of the primer extension product to form amobility-modified primer extension product having a distinctive ratio ofcharge to translation frictional drag. The resulting product may alsocontain a detectable label, which may be incorporated into one or moreof the mobility-modified sequence-specific primer, deoxyribonucleotidesubstrate(s). The detectable label, may be, but is not limited to, aradioactive label or a fluorescent label.

The invention also includes kits useful for carrying out the methods ofthe present invention. Kits of the invention comprise one or moremobility-modified sequence-specific nucleobase polymers. The kits mayalso comprise a second nucleobase polymer, typically an oligonucleotide,which is optionally mobility-modified, where the intended assay requiresa second oligonucleotide; for example, kits for oligonucleotide ligationassays and PCR analysis. Similarly, kits designed for ligase chainreaction amplification will further comprise at least two additionalnucleobase polymers, which together are complementary to a diagnosticligase reaction product. The kits further may also comprise treatingreagents such as restriction enzymes, DNA polymerases, RNases, mismatchbinding proteins, ligases, and exonucleases. Primer extension kitsappropriate for sequencing or oligonucleotide extension assays fordetecting single nucleotide polymorphisms, may further comprisenucleoside triphosphates and/or chain terminating nucleotides.Therefore, components of the kits of the present invention include onemore sequence-specific nucleobase polymers, one or moremobility-modified sequence-specific nucleobase polymers, and/or one ormore nucleoside triphosphates and/or chain terminating nucleotides,wherein one or more of these components may comprise a reporter label.The kit may also comprise reaction buffers for carrying outhybridizations and enzymatic treatments.

In another embodiment, the invention includes kits comprising one ormore of the mobility-modifying phosphoramidite reagents of presentinvention. One or more of the mobility-modifying phosphoramiditereagents, in such kits, may further comprise one or more protectinggroups, reporter molecules, or ligands. Such kits may also comprise oneor more solvents, reagents, or solid surface-bound nucleobase monomerfor use in the synthesis of mobility-modified sequence specificnucleobase polymers.

The mobility-modified nucleobase polymers of the present inventionprovide one or more advantages over currently available modifiedoligonucleotides, as follows. For example, synthesis of themobility-modified nucleobase polymers of the present invention iscompatible with reagents and methods employed in conventional automatedinstruments for DNA synthesis. Furthermore, when the mobility-modifiednucleobase polymers include only uncharged phosphate triester linkages,such as nucleobase polymers according to structural formulae (II) and(III), substantially greater alterations of electrophoretic mobilitiescan be achieved as compared with the charged PEO modifiers in currentuse. As illustrated in the working examples provided infra, there is alarge difference in the electrophoretic mobilities betweenmobility-modified nucleobase polymers of the invention which differ byonly a single mobility-modifying monomeric unit. As a consequence, theinvention permits for greater mobility modifications than can beachieved using conventional PEO modifiers. Significantly,electrophoretic mobility retardations of greater than 100 nucleotidescan be readily achieved using standard DNA and RNA chemistries.

Moreover, use of charged phosphate diester linkages in combination withuncharged phosphate triester linkages, such as the mobility-modifiednucleobase polymers according to structural formulae (II) and (III),greatly increases the repertoire of available, resolvable mobilitymodifications. Thus, the present invention enables the ability tosimultaneously analyze for greater numbers of target nucleic acidsequences than can be analyzed using currently available PEO modifiers.

4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides nucleobase polymer functionalizingreagents and methods for the synthesis of nucleobase polymerfunctionalizing reagents, as well as procedures for the polymerizationof such functionalizing reagents and for their attachment to nucleobasepolymers.

The present invention also relates to novel mobility-modifiedsequence-specific nucleobase polymers that comprise at least onemobility-modifying polymer attached to a nucleobase polymer through oneor more mobility-modifying polymer subunits connected through unchargedlinkages. The mobility-modified sequence-specific nucleic acids andnucleobase polymers provide improved ratios of charge to translationalfrictional drag, allowing more effective electrophoretic separation ofindividual nucleobase polymers within a larger population of nucleicacids, in both sieving and non-sieving electrophoretic media.

The present invention also provides methods for the detection ofnucleotide sequences within one or more target nucleic acids using themobility-modified, sequence-specific nucleobase polymers disclosedherein.

4.1 Abbreviations and Conventions

The abbreviations used throughout the specification and in the FIGS. torefer to the naturally occurring encoding nucleobases are conventionaland are as follows: adenine (A), guanine (G), cytosine (C), thymine (T)and uracil (U).

Unless specified otherwise, nucleobase polymer sequences and/or targetnucleic acid sequences that are represented as a series of one-letterabbreviations are presented in the 5′→3′ direction.

4.2 Definitions

As used herein, the following terms are intended to have the followingmeanings:

“Reporter label,” “reporter label” “label” or “tag:” refers to afluorophore, chromophore, radioisotope, chemiluminescent, spin label, oran enzyme, which causes a detectable event or which allows directdetection of a labeled nucleobase polymer probe by a suitable detector,or a ligand or other first member of a cognate binding pair that canbind specifically and with high affinity to a detectable anti-ligand,anti-hapten, or other second member of a cognate binding pair, such as,but not limited to, reporter-labeled avidin or a reporter-labeledantibody.

“Spectrally resolvable:” means, in reference to a set of fluorescentdyes, that the fluorescence emission bands of the respective dyes aresufficiently distinct, i.e., sufficiently non-overlapping, that thedyes, either alone or when conjugated to other molecules or substances,are distinguishable from one another on the basis of their fluorescencesignals using standard photodetection systems such as photodetectorsemploying a series of band pass filters and photomultiplier tubes,charged coupled devices (CCD), spectrographs, etc., as exemplified bythe systems described in U.S. Pat. Nos. 4,230,558 and 4,811,218 or inWheeless et al., 1985, Flow Cytometry: Instrumentation and DataAnalysis, pp 21-76, Academic Press, New York. Generally, all of the dyescomprising a spectrally resolvable set of dyes are excitable by a singlelight source.

“Nucleobase:” refers to a substituted or unsubstitutednitrogen-containing parent heteroaromatic ring of a type that iscommonly found in nucleic acids. Typically, but not necessarily, thenucleobase is capable of forming Watson-Crick and/or Hoogsteen hydrogenbonds with an appropriately complementary nucleobase. The nucleobasesmay be naturally occurring, such as the naturally-occurring encodingnucleobases A, G, C, T and U, or they may be modified or synthetic.Common modified or synthetic nucleobases include 3-methyluracil,5,6-dihydrouracil, 4-thiouracil, 5-bromouracil, 5-chlorouracil,5-iodouracil, 6-dimethyl amino purine, 6-methyl amino purine, 2-aminopurine, 2,6-diamino purine, 6-amino-8-bromo purine, inosine, 5-methylcytosine, 7-deazaadenine, and 7-deazaguanosine. Additional non-limitingexamples of modified or synthetic nucleobases of which the targetnucleic acid may be composed can be found in Fasman, CRC PracticalHandbook of Biochemistry and Molecular Biology, 1985, pp. 385-392;Beilstein's Handbuch der Organischen Chemie, Springer Verlag, Berlin andChemical Abstracts, all of which provide references to publicationsdescribing the structures, properties and preparation of suchnucleobases.

As will be recognized by those of skill in the art, many of theabove-described modified or synthetic nucleobases are capable of formingWatson-Crick base pairing interactions with the naturally occurringencoding nucleobases A, T, C, G and U. However, in certain embodimentsof the invention, it may be desirable to include in a nucleobase polymersynthetic nucleobases which are not capable of forming Watson-Crick basepairs with either the naturally occurring encoding nucleobases A, T, C,G, and U and/or common analogs thereof, but that are capable of formingnon-standard (i.e., non-Watson-Crick) base pairs with one another.Nucleobases having these properties are referred to herein as“non-standard synthetic” nucleobases. Examples of such non-standardsynthetic nucleobases include, but are not limited to, iso-guanine(iso-G), iso-cytosine (iso-C), xanthine (X), kappa (K), nucleobase H,nucleobase J, nucleobase M and nucleobase N (see U.S. Pat. No.6,001,983). These non-standard synthetic nucleobases base-pair with oneanother to form the following non-standard base pairs: iso-C•iso-G, K•X,H•J and M•N. Each of these non-standard base pairs has three hydrogenbonds. Additional non-standard synthetic nucleobases, as well as methodsfor their synthesis and methods for incorporating them into nucleobasepolymers are found in U.S. Pat. Nos. 5,432,272, 5,965,364 and 6,001,983,the disclosures of which are incorporated herein by reference.

“Nucleobase polymer:” refers to a series of nucleobases that areconnected to one another by linkages that permit the linked polymer tohybridize by standard Watson-Crick base pairs or non-standard base pairsto a target nucleic acid having the complementary sequence ofnucleobases, or that can hybridize to a duplex target nucleic acid toform a triplex structure via Hoogsteen base pairing rules. A variety ofnucleobase polymers capable of hybridizing to a complementary nucleicacid are described in the art. All of these nucleobase polymers arewithin the scope of the invention. Examples of such nucleobase polymersinclude native DNAs and RNAs, as well as analogs of DNAs and RNAs.Common analogs include, but are not limited, to DNAs and RNAs in whichthe respective 2′-deoxyribo- or ribo-nucleosides are connected byphosphonate linkages, phosphoramidate linkages, phosphorothioatelinkages, phosphate triester linkages. Nucleobase polymers also includemolecules having positively charged sugar-guanidyl interlinkages, suchas those described in U.S. Pat. Nos. 6,013,785 and 5,696,253 (see also,Dagani, 1995, Chem. & Eng. News 4-5:1153; Dempey et al., 1995, J. Am.Chem. Soc. 117:6140-6141). Sugar-guanidyl analogs in which the sugar is2′-deoxyribose are referred to as “DNGs,” whereas those in which thesugar is ribose are referred to as “RNGs.” Examples of nucleobasepolymers having a positively charged polyamide backbone with alkylamineside chains are described in U.S. Pat. Nos. 5,786,461; 5,766,855;5,719,262; 5,539,082 and WO 98/03542 (see also, Haaima et al., 1996,Angewandte Chemie Int'l Ed. in English 35:1939-1942; Lesnik et al.,1997, Nucleosid. Nucleotid. 16:1775-1779; D'Costa et al., 1999, Org.Lett. 1:1513-1516 see also Nielson, 1999, Curr. Opin. Biotechnol.10:71-75).

Nucleobase polymers having uncharged backbones have also been describedin the art. For example, nucleobase polymers having uncharged polyamidebackbones are described in WO 92/20702 and U.S. Pat. No. 5,539,082.Nucleobase polymers having uncharged morpholino-phosphoramidatebackbones are described in U.S. Pat. Nos. 5,698,685, 5,470,974,5,378,841 and 5,185,144 (see also, Wages et al., 1997, BioTechniques23:1116-1121).

Additional nucleobase interlinkages which may comprise a nucleobasepolymer include, but are not limited to, peptide-based nucleic acidmimetic interlinkages (see, e.g., U.S. Pat. No. 5,698,685), carbamateinterlinkages (see, e.g., Stirchak & Summerton, 1987, J. Org. Chem.52:4202), amide interlinkages (see, e.g., Lebreton, 1994, Synlett.February, 1994:137), methylhydroxyl amine interlinkages (see, e.g.,Vasseur et al., 1992, J. Am. Chem. Soc. 114:4006), 3′-thioformacetalinterlinkages (see, e.g., Jones et al., 1993, J. Org. Chem. 58:2983),sulfamate interlinkages (see, e.g., U.S. Pat. No. 5,470,967), andlinkages including locked nucleoside analogs (LNA), which includebicyclic and tricyclic nucleoside and nucleotide analogs that may beincorporated into nucleobase polymers that are capable of formingsequence-specific duplex and triplex structures with single stranded anddouble stranded nucleic acids (see, e.g., WO 99/14226).

The nucleobase polymers may be composed wholly of a single type ofinterlinkage, or may comprise combinations of different interlinkages.In certain embodiments, the nucleobase polymer will be a native DNA orRNA, or a common analog thereof.

“Alkyl:” refers to a saturated or unsaturated, branched, straight-chainor cyclic monovalent hydrocarbon group derived by the removal of onehydrogen atom from a single carbon atom of a parent alkane, alkene oralkyne. Typical alkyl groups include, but are not limited to, methyl;ethyls such as ethanyl, ethenyl, ethynyl; propyls such as propan-1-yl,propan-2-yl, cyclopropan-1-yl, prop-1-en-1-yl, prop-1-en-2-yl,prop-2-en-1-yl, cycloprop-1-en-1-yl; cycloprop-2-en-1-yl,prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butyls such as butan-1-yl,butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl,but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl,but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like. Wherespecific levels of saturation are intended, the nomenclature “alkanyl,”“alkenyl” and/or “alkynyl” is used. In certain embodiments, the alkylgroups are (C₁-C₆) linear alkyl groups. Furthermore, as used herein, theterm “lower alkyl” refers to alkyl groups that consist of from one tosix carbon atoms, and, in certain embodiments, lower alkyl groups are(C₁-C₆) linear alkyl.

“Aryl:” refers to a monovalent aromatic hydrocarbon group derived by theremoval of one hydrogen atom from a single carbon atom of a parentaromatic ring system. Typical aryl groups include, but are not limitedto, groups derived from aceanthrylene, acenaphthylene,acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene,fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene,s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene,ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene,phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene,rubicene, triphenylene, trinaphthalene, and the like. In certainembodiments, the aryl group is (C₅-C₁₄) aryl, and, in certainembodiments (C₅-C₁₀). In further embodiments, aryls arecyclopentadienyl, phenyl and naphthyl.

“Capillary electrophoresis:” means electrophoresis in a capillary tubeor channel, where the largest inner dimension of the channelcross-section, which need not be a circle, is between about 25-500microns, allowing efficient heat dissipation throughout the separationmedium, with consequently low thermal convection within the medium.

“Sieving matrix or sieving medium”: refers to an electrophoresis mediumcontaining crosslinks or non-crosslinked polymers which create a networkeffective to retard migration of charged species in an electric field.Examples of a sieving matrix are those based on cross-linkedpolyacrylamide or agarose. A sieving medium may also comprise, asdisclosed in U.S. Pat. No. 5,567,292, polylactams such aspolyvinylpyrrolidone, N,N-disubstituted polyacrylamides andN-substituted polyacrlyamides.

“Non-sieving matrix or non-sieving medium:” refers to a liquid mediumwhich is substantially free of a mesh or network of polymers which areeffective to retard the mobility of analytes.

“Distinctive electrophoretic mobility:” refers to the rate at which ananalyte migrates in an electric field in a particular electrophoreticmedium. A distinctive mobility refers to a distinctive electrophoreticmobility of the analyte, as compared with electrophoretic mobility ofall other detectable analytes present in the sample tested.

“Polymorphism or polymorphic sequences:” refers to a sequence present ina population which shows variation between members of the population.For example, the polymorphisms may relate to single nucleotidedifferences (single nucleotide polymorphisms: SNP) or differences is thenumber of repeat sequences.

“Endonuclease:” refers to any enzyme which cleaves a nucleic acidinternally. An endonuclease may act on either single stranded or doublestranded nucleic acids.

“Short tandem nucleotide repeats:” refers to the collection ofdifferent, simple tandem repeats that are present throughout the genomeof many organisms. In a non-limiting example, one set of repeatedsequences has been characterized as those sequences having the generalformula (A_(w)G_(n)T_(y)C_(n))_(n) where A, G, T, and C represent thefour nucleotides and w, x, y, and z represent number from 0 to 7,wherein the sum of w+x+y+z ranges from 3 to 7 and n is the repeat number(Edwards, A. et al, DNA typing and genetic mapping with trimeric andtetrameric tandem repeats, Am. J. Hum. Genet. (1991) 49(4): 746-56;Caskey, C. T. et al., U.S. Pat. No. 5,364,759).

“Ligand:” refers to a chemical moiety or structure corresponding to onemember of a cognate binding pair that is specifically recognized andbound in a stable complex by a second member of the cognate bindingpair. Examples of such cognate binding pairs include, but are notlimited to, biotin-avidin, and biotin-streptavidin. Other examplesinclude phenyl boronic acid reagents and phenyl boronic acid complexingreagents derived from aminosalicylic acid (see e.g. U.S. Pat. No.5,594,151). Therefore, as used herein, the term ligand encompasses theterm hapten, which refers to a chemical moiety or structure, for exampledigoxigenin, as one member of a cognate binding pair, where the secondmember of the cognate binding pair is an element of the immune system,including but not limited to an intact antibody, a single chainantibody, or an antibody fragment.

“Translational frictional drag:” refers to the measure of a polymer'sfrictional drag as it moves through a defined, sieving or non-sievingmedium.

“Distinctive ratio of charge to translational frictional drag:” refersto the distinctive electrophoretic mobility of a mobility-modifiednucleobase polymer as compared to other detectable nucleic acids ornucleobase polymers, both modified and unmodified, that are presentwithin the sample analyzed.

4.3 Mobility-modifying Phosphoramidite Reagents

In one embodiment, the present invention provides mobility-modifyingphosphoramidite reagents that are nucleobase polymer functionalizingreagents having a structure according to Formula (I):

wherein:

R⁵ is selected from the group consisting of hydrogen, protecting group,reporter molecule, and ligand;

X is selected from the group consisting of O, S, NH, and NH—C(O);

each a is independently an integer from 1 to 6; and

b is an integer from 0 to 40;

R⁶ and R⁷ are each independently selected from the group consisting ofC₁-C₆ alkyl, C₃-C₁₀ cycloalkyl, C₆-C₂₀ aryl, and C₂₀-C₂₇ arylalkyl; and

R² is selected from the group consisting of alkyl comprising at leasttwo carbon atoms, aryl, (R⁸)₃Si— where each R⁸ is independently selectedfrom the group consisting of linear and branched chain alkyl and aryl,base-stable protecting groups, and R⁵—X—[(CH₂)_(a)—O]_(b)—(CH₂)_(a)—.

In certain embodiments, R⁶ and R⁷ are both isopropyl. Where dimersand/or polymers of the mobility-modifying phosphoramidite reagent aredesired, R⁵ is, inter alia, H or other reactive moiety. Moreover, suchdimers and/or polymers can comprise the reagents of the presentinvention either alone or with other mobility-modifying phosphoramiditereagents of the art. In many embodiments, R⁵ is not hydrogen, and inmany embodiments R⁵ is a protecting group. When R⁵ is a protectinggroup, for example, dimers or polymers of the mobility-modifyingphosphoramidite reagent are formed by sequential addition ofmobility-modifying phosphoramidite reagent monomers using standardphosphoramidite synthesis chemistry. As used herein, the phraseprotecting group encompasses not only the conventional, versatile,selectively cleavable protecting groups well known and widely used inphosphoramidite chemistry and as disclosed below, but also thosealternative protecting groups that are not readily or selectivelyremoved by the procedures and conditions of phosphoramidite chemistry.Such alternative protecting groups, particularly those groups resistantto removal under basic conditions, are used in certain embodiments ofthe present invention. For example, in certain embodiments, analternative protecting group, R⁵ is alkyl or other non-readily ornon-selectively cleavable moiety, including, as non-liming examples,compounds of the formulaCH₃—(CH₂)_(d)—O—[—(CH₂)_(a)—O—]_(b)—(CH₂)_(a)—O—, in which a, and b areas defined in Formula (I) above, and d is an integer in the range of 0to 5.

The mobility-modifying phosphoramidite reagents of the present inventionare generally synthesized according to Scheme I, using methods andreagents well known to those skilled in the art, which provides, forexample the illustrative mobility-modifying phosphoramidite reagent ofthe invention (7). In Scheme (I), DMT represents dimethoxytrityl, iPrrepresents isopropyl and R² and b are as previously defined forstructural Formula (I).

Referring to Scheme (I), a DMT protected polyethylene oxidephosphoramidite reagent 7 that can be used in connection with standardphosphoramidite DNA chemistry is prepared.

Initially, phosphorous trichloride 1 is reacted with diisopropylamine 2in a solvent, for example toluene, to formbis(diisopropylamino)chlorophosphine ester 3. Subsequently, thetetraisopropylaminophosphine 3 is reacted with the hydroxyl group onalcohol 4 thereby generating R²-bis(diisopropylamino)phosphite ester 5.Addition of an appropriately protected mobility-modifying polymer havinga free hydroxyl, such as DMT protected polyethylene oxide 6, along withan activator, for example tetrazole, gives rise to the phosphoramidite,reagent 7, an R²-diisopropylaminophosphite ester wherein the polymer isattached to the phosphorous atom through an ester bond.

The phosphoramidite reagent 7 is suitable for use in DNA synthesizers tocouple the mobility-modifying polymer to a nascent nucleobase polymer.As described in Example 2 and illustrated in Scheme (II), infra.

The mobility-modifying phosphoramidite reagents of the present inventionare compatible with standard phosphoramidite synthetic schemes, and,therefore can be used with commercially available instruments fornucleobase polymer synthesis. In addition he mobility-modifyingphosphoramidite reagents of the present invention are readily attachedto either the 5′-end or the 3′-end of a nucleobase polymer.

In one embodiment of the present invention, the mobility-modifyingphosphoramidite reagent of the present invention is added to the 5′-endof a nucleobase polymer attached to a solid support (8), as depicted inScheme II, infra. In this instance the illustrative mobility-modifyingphosphoramidite reagent of the present invention (7) is condensed withthe free 5′-hydroxyl moiety of the surface-bound nucleobase polymer toyield the intermediate structure (9), which is then sequentiallydeprotected to remove base-labile and acid-labile protecting groups andto cleave the product from the solid support, yielding themobility-modified nucleobase polymer (12). In this embodiment, theesterified moiety R² is stable to each step in the above reactionscheme, and, therefore the phosphate triester linkage is uncharged.Moreover, as would be apparent to those skilled in the art, thesurface-bound intermediate (10), can be treated with mild acid, e.g. 3%dichlroacetic acid (DCA), to remove the DMT protecting group to providea free hydroxyl moiety and then condensed with (7) to provide amobility-modified nucleobase polymer with two molecules of (7) joined tothe 5′-end of the mobility-modified nucleobase polymer. Therefore,repetition of this condensation reaction with (7) through n cycles ofreagent addition, provides a mobility-modified nucleobase polymer with nmolecules of the mobility-modifying phosphoramidite reagent of thepresent invention attached to the 5′-end of the nucleobase polymer.

In another embodiment, the mobility-modifying phosphoramidite reagentsof the present invention are added to the 3′-end of a nucleobasepolymer, rather than, or in addition to, the 5′-end of the nucleobasepolymer to be modified. In one illustrative example of this embodiment,depicted in Scheme V, infra. In this synthetic scheme, amobility-modifying phosphoramidite reagent of the present invention (7),is condensed with the 5′-hydroxyl moiety of a nucleobase residue,thymidine, which is attached to a solid support, via, the 3′-hydroxylmoiety of the nucleobase residue. The condensation product (23) obtainedis then oxidized to the phosphate triester (24), deprotected with mildacid to provide the free hydroxyl moiety of (25). The phosphatetriesters synthesized are chiral compounds and both the R and Senantiomers are formed when synthesized according to the methodsdisclosed herein. The racemic mixture of the phosphate triesterssynthesized is used without separation into enantiometrically pure R andS forms. Condensation of (25) with a 5′-protected nucleotidephosphoramidite reagent (26), yields a dinucleobase intermediate havinga mobility-modifying phosphoramidite reagent of the present inventionpositioned between the two nucleobase monomers (27). Repeated cycles ofcondensation with 5′-protected nucleotide phosphoramidite, followed bydeprotection and cleavage from the solid support, provides a nucleobasepolymer carrying a mobility-modifying polymer at the 3′-end of thatnucleobase polymer (29). As noted supra, one or more of themobility-modifying phosphoramidite reagents of the present invention canbe added to the nucleobase polymer either alone or in combination withone or more mobility-modifying phosphoramidite reagents known in theart. Accordingly, one or more of the linking groups formed betweenmobility-modifying phosphoramidite reagents or between amobility-modifying phosphoramidite reagent and the nucleobase polymercan be a charged phosphate diester bond. Furthermore, one or more of themobility-modifying reagents employed can be a branched compound, e.g. asin structure (21), provided it is not the 5′-proximal moiety with whicha 5′-protected nucleotide phosphoramidite is to be condensed.

In another embodiment a mobility-modifying phosphoramidite reagent ofthe present invention is attached to a readily-cleaved chemical linkthat is bonded to a solid support such as controlled pore glass orpolystyrene. An example of such a readily-cleaved chemical link bondedto a solid support is compound 33:

the synthesis of 33 is described below:

In this manner, nucleobase polymers carrying mobility-modifying reagentsat the 3′-end are assembled without the additional nucleobase residue atthe 3′-end of the molecule, as depicted in Scheme V and compound (29).However, the advantage of the general method depicted in Scheme V, isthat nucleobase-bound solid supports not only are commerciallyavailable, but they are also provided in the form of pre-assembledcartridges, which are filled with a nucleotide-bound solid support, thatare compatible with automated nucleobase polymer synthesizing machines.

The mobility-modifying phosphoramidite reagents of the present inventionyield uncharged phosphate triester linkages when joined to either orboth of the 5′-end and/or the 3′-end of a nucleobase polymer, as well aswhen joined to another mobility-modifying phosphoramidite reagent of thepresent invention. Accordingly the esterified moiety, i.e. R² of FormulaI, and, in certain embodiments, R¹⁰ and/or R⁴ of Formulae II and III,infra, will be stable to all steps of conventional phosphoramiditechemistry. That is the esterified moiety should be not be removedduring, inter alia, the deprotection steps, depicted, for example inSchemes III and V. More specifically, the esterified moiety should bestable to the procedures and conditions required for deprotection ofprotected amines and cleavage of the mobility-modified nucleobasepolymer from the solid support, such that the linkage between themobility-modifying monomer units and between a mobility-modifyingmonomer unit and the 3′-end or 5′-end of the nucleobase polymer is anuncharged phosphate ester.

Accordingly, when R² of Formula I, and R¹⁰ and/or R⁴ of Formulae II andIII, infra, are alkyl, R², and R¹⁰ and/or R⁴ are selected from the groupconsisting of alkyl comprising at least two carbon atoms, e.g. C₂-C₆linear alkyl.

The mobility-modifying phosphoramidite reagents of the present inventionmay also be used in conjunction with other mobility-modifyingphosphoramidite reagents that are known in the art, including, forexample, the representative PEO phosphoramidite reagent depicted supra,which comprises an esterified cyanoethyl moiety. As depicted in SchemeIII, infra, the surface-bound nucleobase polymer intermediate (10),which comprises one molecule of a mobility-modifying phosphoramiditereagent of the present invention, can be treated with mild acid, e.g. 3%DCA, to remove the DMT protecting group to provide the surface-boundcompound (13) carrying a free hydroxyl moiety. Condensation of the PEOphosphoramidite reagent, (14), with surface-bound (13) provides anucleobase polymer with two mobility-modifying reagents joined to the5′-end of the nucleobase polymer. In this scheme, deprotection andcleavage of the product from the solid surface also results in removalof the esterified cyanoethyl group of reagent (14). Therefore, in thisembodiment, the linkage formed between the second mobility-modifyingreagent (14) added and the mobility-modified nucleobase polymer, is acharged phosphate diester bond.

In a further embodiment, the mobility-modifying phosphoramidite reagentsof the present invention encompass dendritic reagents comprising twoesterified mobility-modifying groups attached to each phosphorous atom.An example of such a compound (21) and a general scheme for itssynthesis are depicted in Scheme IV, infra. As would be apparent tothose skilled in the art, repeated cycles of condensation using reagent(21) would provide a geometric increase in the number ofmobility-modifying phosphoramidite reagents attached to a surface-boundnucleobase polymer, according to the synthetic method depicted in SchemeIV, infra. It would also be apparent to one of skill in the art that anucleobase polymer can be modified with one or more of themobility-modifying phosphoramidite reagents (7), (14), and (21),generally according to Scheme II, III, or V, infra, to provide a seriesof mobility-modified nucleobases polymers, having one or more branchedand/or unbranched, charged and/or uncharged mobility-modifying moieties.

4.4 Compositions of Mobility-modified Nucleobase Polymers

In one aspect of the present invention, mobility-modifying polymerchains are attached to sequence-specific nucleobase polymers by alinking group. Various polymers adaptable to the embodiment include,among others, polyoxides, polyamides, polyimines, and polysaccharides.The compositions also embody polymer chains in the form of copolymers orblock polymers, such as polyethylene oxide and polyamine (see e.g.Vinogradov, S. V. (1998) Self assembly of polyamine-poly(ethyleneglycol) copolymers with phosphorothioate nucleobase polymers,Bioconjugate Chem., 9: 805-12), having one or more uncharged linkersbetween monomer units.

In one embodiment, mobility-modifying polymers are polyoxides orpolyethers. In this context, the term polyoxide is used to denotepolymers with oxygen atoms in the main chain, particularly those withmonomer units of the type [O—(CH₂)_(n)] where n is an integer selectedfrom the range of 1 to 15, in certain embodiments n is selected from therange of 2 to 6, and in other embodiments, n=2, together with theirderivatives. Linear polyoxides applicable to the composition include,for example, poly(methylene oxide), poly(ethylene oxide),poly(trimethylene oxide) and poly(tetramethylene oxide). Branchedpolyoxides provide additional bases for mobility-modification by, insome cases, imparting to the mobility-modified nucleobase polymer atranslational frictional drag that is different than that provided by alinear polymer chain. Branched polymers, for example poly(propyleneoxide) which are appreciably soluble in aqueous solvents, are used incertain embodiments. Other applicable branched polymers includepoly(acetaldehyde), and poly(but-1-ene oxide).

In another embodiment, the mobility-modifying polymer is a monodisperselinear polyoxide of polyethyleneoxide (PEO) because of its high degreeof solubility in a variety of aqueous and organic solvents. Moreover,chemistry of poly(ethylene oxide) and methods of use for modifyingchemical and biological compounds are well known in the art. (see e.g.Grossman, P. D. et al., U.S. Pat. No. 5,777,096; Muller, W. et al.(1981) Polyethylene glycol derivatives of base and sequence-specific DNAligands: DNA interaction and application for base specific separation ofDNA fragments by gel electrophoresis, Nucleic Acids Res. 9: 95-119);Maskos, U., (1992) Oligonucleotide hybridization on glass supports: anovel linker for oligonucleotide synthesis and hybridization propertiesof oligonucleotides synthesized in situ, Nucleic Acids Res. 20:1679-84).Accordingly, those skilled in the art can readily vary the number ofpolyethylene units in the mobility-modifying polymer to impartdistinctive ratio of charge to translational frictional drag to themobility-modified sequence-specific nucleobase polymer.

In addition, the mobility-modifying polymers of the embodiment mayfurther comprise functional groups, such as a hydroxyl, sulfhydral,amino or amide group. These functional groups permit attachment ofvarious reporter molecules, ligands, or other polymer chains. Protectinggroups may be present on the functional group when themobility-modifying polymer is being coupled to the sequence-specificnucleobase polymer, or during reaction of other functional groups withthe sequence-specific mobility-modified nucleobase polymer. Groupssuitable for protecting specific functional groups, including methodsfor removal, are well known in the art such that the art provides ampleguidance for selecting the appropriate protecting reagents (see e.g.Greene and Wuts, Protective Groups in Organic Synthesis, 2nd ed., JohnWiley & Sons, Inc., New York, 1991). For example, hydroxyl groups areprotectable with acid labile groups such as dimethoxytrityl (DMT), orwith base labile group such as fluorenyl methyl chloroformate (Fmoc).

Protecting groups useful in the present invention, encompass not onlythe conventional, versatile, selectively cleavable protecting groupswell known and widely used in phosphoramidite chemistry and as disclosedabove, but also those alternative protecting groups that are not readilyor selectively removed by the procedures and conditions ofphosphoramidite chemistry. Such alternative protecting groups,particularly those groups resistant to removal under basic conditions,are used in certain embodiments of the present invention. For example,in certain embodiments, as an alternative protecting group, R⁵ is alkylor other non-readily or non-selectively cleavable moiety, including, asnon-liming examples, compounds of the formulaCH₃—(CH₂)_(c)—O—[—(CH₂)_(a)—O—]_(b)—(CH₂)_(a)—O—, in which a, and b areas defined in Formula (I) above, and c is an integer in the range of 0to 5.

Another aspect of the invention involves linking groups that attach themobility-modifying polymer to the sequence-specific nucleobase polymer.In a general embodiment, the group attaching the mobility-modifyingpolymer chain to the nucleobase polymer comprises phosphate triester,phosphonate, phosphoamidate, phosphothioester or phosphodithioatelinkage. Phosphonate and phosphate triester linkages permit attachmentof other chemical constituents to the phosphorous atom to effect furtherdifferences in the ratio of charge to translational frictional dragbetween mobility-modified nucleobase polymers. Thus, one embodimentincludes alkylphosphonate linkages, such a methyl phosphonate.

In a further embodiment, the linkage is a phosphate triester, whereinthe free ester has attached various chemical groups so as to render thelinker uncharged, such as alkyls, functionalized alkyls, or polymers.When the chemical group is an alkyl, the compound may be a linear orbranched alkyl, generally a lower alkyl group. Linear alkyls include,but are not limited to, methyl, ethyl, propyl, or butyl groups, whilebranched alkyls include, but are not limited to, isopropyl or tertbutylgroups. However the chemical groups attached to the free ester aregenerally limited to those groups that are stable to all steps ofconventional phosphoramidite chemistry, including deprotection steps andespecially to the procedures and conditions required for thedeprotection of protected amines, such that the resulting linkage is anuncharged phosphate triester. Therefore, when such groups are alkyl, thegroup is generally an alkyl other than methyl, for example, C₂-C₆ linearalkyl, since mono methyl phosphate triesters tend to be less stable thanhigher-order alkyl phosphate triesters. The alkyl group may also haveattached functional moieties, such as reporters, ligands or biotinmolecules. Reporter molecules include but are not limited tofluorescent, chemiluminescent or bioluminescent molecules, while ligandsinclude, but are not limited to, molecules such as cholesteryl,digoxigenin, 2,4 dinitrophenol, and biotin. When the chemical group is apolymer, the same types of polymers set forth above, including but notlimited to polyoxides, polyamides, polyimines, polysaccharides, andpolyurethanes, function as suitable substituents.

The sequence-specific nucleobase polymers in one embodiment of theinvention are natural or synthetic. Natural nucleic acids andoligonucleotides are obtained by cloning the desired fragment in acloning vector and isolating the desired nucleic acid fragment byrestriction enzyme digestion. Alternatively, they are made usingoligonucleotide templates and enzymatic synthesis, such as polymerasechain reaction (Sambrook, J. et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, New York, 2000).

In another embodiment, the sequence-specific nucleobase polymers aresynthetic nucleobase polymers. Synthetic sequence-specific nucleobasepolymers comprise deoxyribonucleic acids (DNA), ribonucleic acids (RNA)or composites of DNA and RNA. Further modification of the nucleobases ofthe deoxy or ribonucleic acids are possible. Modified bases includeinosine, deoxynapthosine, etheno adenosine and cytidine, andbromodeoxyuridine. Other modified bases readily used in nucleobasepolymer synthesis are 7-deaza purines, N⁶ methyl adenosine, O⁶ methylguano sine, and 2-aminopurine.

In another embodiment, the sequence-specific nucleic acids andnucleobase polymers are analogs, for example phosphonate nucleobasepolymer, nucleobase polymers comprising one or more locked nucleosideanalogues, peptide nucleic acids (PNA), phosphorothioate nucleobasepolymers, phosphate triester nucleobase polymers, or nucleobase polymershaving chain terminating nucleosides.

Phosphonate nucleobase polymers have a backbone comprising phosphonateinternucleotide linkages. The phosphonates known in the art ofnucleobase polymer synthesis include H-phosphonate, alkyl phosphonate(e.g. methylphosphonate), 2-aminoethylphosphonate, and benzylphosphonate(Samstag, W., (1996) Synthesis and properties of new antisensenucleobase polymers containing benzylphosphonate linkages, AntisenseNucleic Acid Drug Dev. 6: 153-56; Fathi, R., (1994) nucleobase polymerswith novel, cationic backbone constituents: aminoethylphosphonate,Nucleic Acids Res. 22: 5416-24; Seliger, H., (1990). Simultaneoussynthesis of multiple nucleobase polymers using nucleoside-H-phosphonateintermediates, DNA Cell Biol., 9: 691-6; Zhou, Y., (1996) Solid-phasesynthesis of oligo-2-pyrimidinone-2′-deoxyribonucleotides andoligo-2-pyrimidione-2′deoxyriboside methylphosphonates, Nucleic AcidsRes. 24: 2652-2659). The advantages of nucleobase polymers havingphosphonate linkages are their property of readily forming stable triplehelix structures and their higher resistance to nuclease action.

Locked nucleoside analogues (LNA) include bicyclic and tricyclicnucleoside and nucleotide analogues that may be incorporated intonucleobase polymers that are capable of forming sequence-specific duplexand triplex structures with single stranded and double stranded nucleicacids. Those duplex and triplex structures that comprise LNA-containing,sequence specific nucleobase polymers are more thermostable than thecorresponding structures formed with non-analogue-containing nucleobasepolymer molecules (see e.g. WO 99/14226).

Peptide nucleic acids (PNA) are synthetic polyamides which compriserepeating units of the amino acid, N-(2-aminoethyl)-glycine to whichbases such as adenine, cytosine, guanine, thymine are attached via themethylene carbonyl group. Other bases including pseudo isocytosine, 5methyl cytosine, pseudouracil, hypoxanthine are suitable forincorporation into PNAs. The resistance of PNA nucleobase polymers tonucleases and the high stability of PNA-DNA hybrids make them desirableprobes for identifying target polynucleotides, except in methodsrequiring nuclease treatments (Egholm, M., et al. (1992) Peptide nucleicacids (PNA): Oligonucleotide analogues with an achiral peptide backbone,J. Am. Chem. Soc. 114: 1895-1876; Hanvey, J. C., (1992) Antisense andantigene properties of peptide nucleic acids, Science 258:1481-5;Nielson, P. E. et al., (1993) Sequence-specific inhibition of DNArestriction enzyme cleavage by PNA nucleic acids, Nucleic Acids Res. 21:197-200).

Oligonucleotide and nucleobase polymer analogs with phosphorothioate orphosphorodithioate internucleotide linkages have sulfur atom in place ofthe oxygen as the non-bridging ligands bound to the phosphorous(Eckstein, F., (1985), Nucleoside phosphorthioates, Ann. Rev. Biochem.,54: 317-402). Phosphorothioate diesters are chiral at the phosphorousatom (Rp and Sp) and have utility as nucleobase polymer probes andantisense molecules because of their resistance to nucleases (Zon, G.,Phosphorothioate oligonucleotides. In Oligonucleotides and Analogues: Apractical approach, (Eckstein, F. ed) IRL Press, Oxford, pg 87-108).Phosphorodithioates have both the non-bridging ligands bound to thephosphorous as sulfur atoms (Caruthers, M. H. et al., (1992), Chemicalsynthesis of Deoxyoligonucleotides and Deoxyoligonucleotide analogs,Meth. Enzy. 211: 3-20).

In another embodiment of the invention, the sequence-specific nucleobasepolymer analogs comprise different combinations of internucleotidelinkages. Thus a nucleobase polymer may comprise methylphosphonate,phosphate diester, and N-(2-aminoethyl)-glycine internucleotide linkages(Miller, P. S. et al., (1999), A psoralen-conjugated triplex formingoligodeoxyribonucleotide containing alternatingmethylphosphonate-phosphate diester linkages: synthesis and interactionswith DNA, Bioconjugate Chem. 10: 572-577; Gildea, B. D. et al., U.S.Pat. No. 6,6063,569). Other sequence-specific nucleobase polymer analogsmay comprise a combination of phosphorothioate-phosphate diesterinternucleotide linkages (see e.g. Ghosh M. K. (1993),Phosphorothioate-phosphate diester oligonucleotide co-polymers:assessment for antisense, Anticancer Drug Des, 8(1): 15-32).Combinations of different internucleotide linkages offer nucleobasepolymers with different hybridization characteristics and nucleaseresistance.

In another embodiment of the present invention, the mobility-modifiedsequence-specific nucleobase polymer comprises at least onenon-negatively charged internucleotide linkage. In one non-limitingexample, at least one internucleotide linkage of the mobility-modifiedsequence-specific nucleobase polymer is an uncharged mono alkylphosphate triester linkage, while in another non-limiting example, theinternucleotide linkage is a positively charged amide linkage comprisingan alkylamine side chain. In this embodiment, the non-negatively chargedinternucleotide linkage further alters the charge to translationalfrictional drag ratio of the mobility-modified sequence-specificnucleobase polymer of the present invention. In another non-limitingexample, at least one internucleotide linkage of the mobility-modifiedsequence-specific nucleobase polymer is a phosphoramidate linkage, whichis another non-negatively charged internucleotide linkage that willalter the charge to translational frictional drag ratio of themobility-modified sequence-specific nucleobase polymer of the presentinvention. Synthesis of oligonucleotides comprising a phrophoramidateinternucleotide linkage is described in U.S. Pat. No. 5,476,925, whichis hereby incorporated by reference in its entirety.

In another embodiment the mobility-modified sequence-specific nucleobasepolymers are conjugated to various reporters, ligands and polymermolecules. All components of the mobility-modified sequence-specificnucleic acid or nucleobase polymer are amenable to conjugation,including at internal or terminal nucleotide residues, the phosphatetriester group linking the polymer to the nucleobase polymer, and themobility-modifying polymer. Those skilled in the art are well versed ingenerating the appropriate modifications to form the desired conjugate(see e.g. Oligonucleotides and Analogs, F. Eckstein, ed., Chapter 8-9,IRL Press, 1990; Agrawal, S. (1986), Efficient methods for attachingnon-radioactive labels to the 5′ ends of syntheticoligodeoxyribonucleotides, Nucleic Acids Res. 14: 6227-45; Nelson,(1992), Nucleic Acids Res., 20 (23): 6253-6259). When the conjugation isto the polymer of the mobility-modified nucleobase polymer, the polymerhas appropriate functional groups such as hydroxyl, sulfhydral, amide oramino groups that permit attachment of one or more reporter, and ligandmolecules. When the conjugation is to a phosphate triester linkinggroup, the free ester provides the site of attachment.

Conjugating various reporter and ligand moieties permits detection,modification, or immobilization of the sequence-specificmobility-modified nucleobase polymer. They also permit furthermobility-modification of the sequence-specific mobility-modifiednucleobase polymer. Reporter molecules include, but are not limited tofluorescent compounds, such as fluoresceine, rhodamine, Texas red,cyanine dyes, and acridine dyes. Ligands include, but are not limitedto, 2,4 dinitrophenol, digoxigenin, and cholesterin, as well as enzymes,or enzyme substrates that could be attached to the sequence-specificmobility-modified nucleobase polymer. Conjugated ligands, including butnot limited to biotin, permit isolation, detection, or immobilization ofthe mobility-modified nucleobase polymer by binding to avidin orstreptavidin.

In view of the embodiments set forth above, certain embodiments ofsequence-specific mobility-modified nucleic acids or nucleobase polymershave a structure according to structural formula (II) or (III):

or a salt thereof, wherein:

R², R⁵, X, a, and b are as in Formula (I);

each R¹⁰ is independently selected from the group consisting of hydrogenand R²;

each R⁴ is independently selected from the group consisting of hydrogenand R²;

each b is independently an integer from 0 to 40;

each d is independently an integer from 1 to 200; and

OLIGO is a sequence-specific nucleobase polymer, typically comprising atleast 5 nucleobases, with the proviso that at least one R¹⁰ or at leastone R⁴ is other than hydrogen.

Amongst the various mobility-modified nucleobase polymers of structuralformulae (II) and (III) are those compounds in which each X is O, each ais the same (generally 2), each b is in the range of 1 to 15 and, and incertain embodiments, b is in the range of 1 to 6, and the OLIGO is aDNA, RNA, and/or an analog of DNA or RNA, oligomer, each d is in therange of 1 to 200, in certain embodiments in the range of 1 to 100, andfurther embodiments in the range of 1 to 50.

In the compounds of structural formula (III) each R¹⁰ and/or R⁴ may,independently of one another, be a hydrogen atom. When R¹⁰ and/or R⁴ arehydrogen, the resultant phosphate ester group typically has a pKa in therange of 0 to 1. Thus, when the pH of the assay conditions is above thepKa, as is usually the case in biological assays, the hydrogen atomexchanges with solvent and at least, a net fraction of the phosphateester groups are negatively charged. Due to the ionizability of thephosphate ester group, those of skill in the art will appreciate thatfor purposes of defining the invention, selecting R¹⁰ and/or R⁴ to behydrogen includes within its scope both the unionized form and theionized (i.e., negatively charged) form of the resultant phosphate estergroup.

Similarly, other groups within the illustrated or described compoundsmay be ionizable. Moreover, many of the compounds may include chiralcenters or exist in different tautomeric or geometric isomeric forms. Asany structural drawings may represent only a single ionizable,tautomeric, enantiomeric or geometric isometric forms, it will beunderstood that the structural drawings are not intended to be limiting,and any non-illustrated ionizable, tautomeric, enantiomeric or geometricisomeric forms of the compounds are intended to be within the scope ofthe present invention.

In the mobility-modified nucleobase polymers of structural formulae (II)and (III), the mobility-modifying segment of the molecule may bebranched or linear. When linear, each R¹⁰ and R⁴ is either hydrogen(where possible), alkyl comprising at least two carbon atoms, or aryl.When branched, at least one R¹⁰ or R⁴ isR⁵—X[(CH₂)_(a)—O]_(b)—(CH₂)_(a)—. In these embodiments where anuncharged phosphate ester linkage is desired, the chemical groupsattached to the free ester are generally limited to those groups thatare stable to all steps of conventional phosphoramidite chemistry,including deprotection steps and especially the procedures and basicconditions required for the deprotection of protected amines. Therefore,when such groups are alkyl, the group is an alkyl other than methyl, forexample, C₂-C₆ linear alkyl, since methyl phophotriesters tend to beless stable than higher-order alkyl phosphate triesters.

Identical or different mobility-modifying polymer combinations can beused to provide the mobility-modified sequence-specific nucleobasepolymer with distinctive, predictable mobility alterations. Thesequence-specific nucleobase polymers comprise DNA, RNA, or analogsthereof, as described above. The OLIGO may be labeled or unlabeled.Those skilled in the art are well versed in devising nucleobase polymersequences useful for detecting a selected nucleic sequence within one ormore target nucleic acids by the various methods described below.

4.5 Methods of Synthesis

The methods for synthesizing the nucleobase polymer-functionalizingreagents of the present invention and for synthesizing the variouscompositions of mobility-modified sequence-specific nucleobase polymerscomprising those functionalizing reagents, follow variations of knownreaction schemes used for sequence-specific nucleobase polymer synthesisand modification. As regards the mobility-modifying polymers of theembodiments, methods of synthesizing various polymers are well known.Polymers suitable as mobility-modifiers include, among others,polyoxide, polyamide, polyimine, and polysaccharides (see e.g. Molyneux,P., Water-Soluble Synthetic Polymers: Properties and Behavior, CRCPress, 1984; Gravert, D. J., (1977) Synthesis on soluble polymers: newreactions and the construction of small molecules, Curr Opin Chem Biol,1 (1):107-13). Long chain alcohols, such as lipohilic C₁₆ alcohols usedas spacer arms to conjugate functional moieties, are also suitable asmobility-modifiers. Functional groups, such as the hydroxyls onpolyoxides, are protected by an appropriate protecting agent, such as4′, 4′-dimethoxytrityl chloride. Groups suitable for protecting specificfunctional groups, as well as methods for removal, are well known andwill be apparent to those skilled in the art. Guidance for selectingprotecting reagents can be found, for example, in Greene and Wuts,Protective Groups in Organic Synthesis, 2nd ed., John Wiley & Sons,Inc., New York, 1991)

The nucleobase polymers attached to the mobility-modifying polymerchains are natural or synthetic nucleic acids having defined nucleotidesequences. In one embodiment the sequence-specific nucleobase polymer isa natural DNA or RNA. Natural nucleic acids are readily prepared bycloning the desired fragment in a cloning vector and isolating thedesired nucleic acid fragment by restriction enzyme digestion of therecombinant molecule. Alternatively, they are made from nucleic acidtemplates by enzymatic synthesis, such as polymerase chain reaction(Sambrook, J. et al., supra).

In one embodiment, the nucleobase polymers are chemically synthesizedusing the phosphoramidite or phosphate triester methods, either insolution or on a suitable inert solid support. Methods for nucleobasepolymer synthesis are well know to those skilled in the art (see e.g.Caruthers et al. (1982) Genetic Engineering 4:1-17; Users Manual Model392 and 394 Polynucleotide Synthesizers, (1990), pages 6-1 through 6-22,Applied Biosystems, Part No. 901237).

The phosphoramidite method is one method of synthesis forsequence-specific oligodeoxyribonucleic and oligoribonucleic acids. Ingeneral, a protected nucleoside is conjugated to a solid support andthen treated with acid, for example trichloroacetic acid, to remove the5′-hydroxyl protecting group, thus generating a free hydroxyl group forthe subsequent coupling reaction. A protected nucleoside phosphoramiditemonomer and an activating reagent such as tetrazole are reacted with thenucleoside bound to the solid support. The activating agent protonatesthe nitrogen of the phosphoramidite, permitting nucleophilic attack ofthe phosphorous atom by the exposed hydroxyl group. Following nucleosideaddition, the growing chain is capped, generally with acetic anhydrideand 1-methylimidazole, to terminate any nucleotide chains that failed toreact. The internucleotide phosphite linkage is oxidized, with iodine asthe preferred oxidizing agent, to the more stable phosphate triester.Following oxidation, the hydroxyl protecting group is removed with aweak acid, such as trichloroacetic and the cycle of reactions isrepeated until formation of a nucleobase polymer of the desired lengthand sequence is complete. Base treatment cleaves the nucleobase polymerfrom the solid support and also removes any phosphate protecting groups,such as β-cyanoethyl. Complete deprotection of the exocyclic aminogroups on the nucleoside bases is accomplished by treating thenucleobase polymer in base at elevated temperatures, for example 55° C.in concentrated ammonium hydroxide. The remaining protecting groups,usually dimethoxytrityl (DMT) groups on the 5′-hydroxyl, are removedduring synthesis or, alternatively, may be left on if reverse phase HPLCis the purification method of choice. After synthesis, the nucleobasepolymer is amenable to labeling at the 5′ terminus (see e.g.Oligonucleotides and Analogs, F. Eckstein (1990), Ed. Chapter 8, IRLPress; Orgel et al., (1983) Nucleic Acids Research 11:6513; U.S. Pat.No. 5,118,800), the phosphate diester internucleotide linkages (seee.g., Orgel et al., supra at Chapter 9), or the 3′ terminus (see e.g.Nelson, (1993), Nucleic Acids Research 20:6253-6259).

In another embodiment, the nucleobase polymers are analogs havingmodifications of the base, the sugar, or the backbone. Modifications inthe backbone, include, but are not limited to polyamide (i.e. peptidenucleic acids), phosphonate, phosphorothioate, phosphodithioester, andphosphoamidate internucleotide linkages. Methods of synthesizingsequence-specific nucleobase polymer analogs with modifiedinternucleotide linkages are well known to those skilled in the art (seee.g. Oligonucleotides and Analogs, A Practical Approach, Eckstein, F.,Ed., IRL Press, (1990); Agrawal, S., (1993), Protocols forOligonucleotides and Analogs, Meth. Mol. Biol., Vol 20, Humana Press).

Sequence-specific nucleobase polymers with polyamide backbones betweennucleobases are also known as peptide nucleic acids (PNA), one exampleof which is a polymer having repeating units of N-(2-aminoethyl)-glycineto which nucleobases are attached through a methylene carbony group(Nielson, P. E. (1991), Sequence-selective recognition of DNA by stranddisplacement with a thymine-substituted polyamide, Science254:14971500).

As used herein, “PNA” refers to a polymer of nucleobases linked togethervia an uncharged polyamide backbone. The PNA backbone may be anybackbone of acyclic, achiral and neutral polyamide linkages to whichnucleobases can be attached and that satisfies the criteria discussedsupra. PNAs useful in the present methods are described, for example, inU.S. Pat. No. 5,539,082 and WO 92/20702, the disclosures of which areincorporated herein by reference. The amino acids which form thepolyamide backbone may be identical or different, but are generallyidentical. In certain embodiments, PNAs are those in which thenucleobases are attached to an N-(2-aminoethyl)-glycine backbone, i.e.,a peptide-like, amide-linked unit (see, e.g., U.S. Pat. No. 5,719,262;Buchardt et al., 1992, WO 92/20702; Nielsen et al., 1991, Science254:1497-1500).

Various strategies for PNA synthesis are available. In one method, PNAmonomers used for synthesis have the exocyclic amino groups protected bybenzyloxycabonyl (Z) while the amine is protected with tertbutyloxycarbonyl (tBoc). Protected monomer bound to a solid substrate isdeprotected using a strong acid, such a trifluoroacetic acid (TFA), togenerate a free amino group for the subsequent coupling to the next PNAmonomer. A PNA monomer activated, for example by carbodiimides or O—(7azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexaflurophosphate(HATU), reacts with the free amino group of the solid support bound PNA,to form the amide bond. Capping of the PNA chain is accomplished withacetic anhydride in presence of N-methylpyrrolidone and pyridine (seee.g. Koch, T., et al. (1997) Improvements in automated PNA synthesisusing Boc/Z monomers, J. Peptide Res. 49:80-88; Maison, W. (1991)Modified PNAs: A simple method for the synthesis of monomeric buildingblocks, Bioorg. Med. Chem. Lett., 9: 581-584). The completed nucleobasepolymer is removed from the solid support with a strong acid, forexample hydrofluoric acid or trifluoromethane sulfonic acid. Otherstrategies for synthesizing PNAs include use of 9-fluorenylmethoxycarbonyl (Fmoc) or monomethoxytrityl protecting groups (Breipohl, G., etal, (1996), Synthesis of polyamide nucleic acids (PNAs) using a novelFmoc/Mmt protecting group combination, Bioorg. Med. Chem. Lett.6:665-670; Will, D. W. et al., The synthesis of polyamide nucleic acidsusing a novel monomethoxytrityl protecting group strategy, Tetrahedron,51:12069-12082). Also available are PNA synthesis methods that use acidlabile backbone protecting groups and base labile protecting groups forthe exocyclic amines on the nucleobases (Gildea, B. D., U.S. Pat. No.6,063,569).

Phosphonate nucleobase polymer analogs have H-phosphonate oralkylphosphonate internucleotide linkages. Nucleoside hydrogenphosphonate monomers used for the synthetic cycle is suitably protectedby protecting groups normally used in the phosphoramidite syntheticscheme. The 5′-OH protecting group on the protected nucleoside bound tothe solid support is removed with acid, such as dichloroacetic acid. Inthe coupling step, the nucleoside phosphonate monomer is activated, forexample by pivaloyl chloride, 1-adamantane carbonyl chloride, ordipentaflurorophenyl carbonate, resulting in formation of an anyhydridethat reacts with the free hydroxyl group on the solid support boundnucleoside. Capping is dependent on the type of activating reagent: useof unhinderd activating reagent, such as paivaloyl chloride, may notrequire capping. Otherwise, capping is carried out with agents such ascyanoethyl-H-phosphonate or isopropyl-H-phosphonate. Unlike thephosphoramidite method set forth above, there is no oxidation of thephosphonate internucleotide linkage. Subsequent base treatment resultsin release of the completed nucleobase polymer from the solid supportand removal of exocyclic protecting groups (see e.g. Seliger, H. (1990),Simultaneous synthesis of multiple oligonucleotides using nucleosideH-phosphonate intermediates, DNA Cell Biol. 9 (9) 691-96.

Nucleobase polymer analogs with alkylphosphonate internucleotidelinkages are readily synthesized by several methods (Oligonucleotidesand Analogues, A Practical Approach, Eckstein, F., Ed., IRL Press, pp137-154). By way of example, one exemplary method usesalkylphosphonamidite chemistry for the synthesis reactions. In thismethod, the nucleoside monomers used for the synthetic reactions havethe exocyclic amines protected with suitable protecting groups, forexample benzoyl, isobutyryl, or tert-butylphenoxyacetyl (t-BPA) groups,while the 5′ hydroxyls are protected with a group such as a pixyl ordimethoxytrityl. (Sinha, N. D. et al. (1994), Synthesis ofoligodeoxynucleoside methylphosphonates utilizing the tertbutylphenoxyacetyl group for exocyclic amine protection, Nucleic Acids.Res. 22 (15): 3119-23; Hogrefe, R. I. et al (1993), An improved methodfor synthesis and deprotection of methylphonate oligonucleotides, Meth.Mol. Biol. 20: 143-63; Zhou, Y. (1996), Solid phase synthesis ofoligo-2-pyrimidone-2″deoxyribonucleotides andoligo-2-pyrimidone-2′-dexoriboside methylphosphonates, Nucleic Acids.Res., 24: 2652-2659). Alkyphosphonamidites nucleoside reagents areprepared by phosphitylation, which involves reactingalkyldichlorophosphine, such a methyldichlorophosphine, anddiisopropylamine with the appropriately protected nucleoside, therebyforming the 5′-protected nucleoside diisopropylmethylphosphonamidite. Inthe nucleobase polymer synthetic cycle, monomer bound to the solidsubstrate is deprotected (i.e. depixylated or detritylated) using acid,for example dichloroacetic acid, and reacted with thealkylphosphonamidite nucleoside reagent in presence of an activatingagent, such a tetrazole. The free OH group attacks the phosphorous atomgenerating a methylphosphonite intermediate. Subsequent oxidation, whichprecedes the capping step, results in conversion to themethylphosphonate linkage. Capping is accomplished with acetic anhydrideand dimethylaminopyridine. Continued cycles of deprotection, coupling,oxidation and capping generate the desired oligonucleotide. Release fromthe solid support is affected by gentle deprotection, for example bytreatment with ethylenediamine or hybrazine hydrate (see e.g. Hogrefe,R. I., An improved method for synthesis and deprotection ofmethylphosphonate oligonucleotides, In Protocols for Oligonucleotidesand Analogs, Meth. Mol. Biol., Vol 20, Sudhir Agrawal, Ed., HumanaPress, Inc. 1993). Other phosphonates, such as benzylphosphonates andaminoethylphosphonates, may also serve as internucleotide linkages(Samstag, W. et al., supra; Fathi, R., et al., supra).

Phosphorothioate and phosphorodithioate sequence nucleobase polymeranalogs have sulphur substituted for oxygen as the non-bridging ligandsbonded to the phosphorous atom. Synthesis of sequence-specificnucleobase polymers having phosphorothioate linkages is accomplished bya variety of methods, including H-phosphonate or phosphoramiditechemistry. One exemplary method uses phosphoramidite route to synthesiswhich permits introduction of the phosphorothioate linkage at any pointin the nucleobase polymer synthetic scheme. In this method, the solidsupport bound nucleoside, which is protected at the 5′ OH group withDMT, is deprotected and coupled to the incoming nucleosidephosphoramidite reagent in the presence of an activating agent, such astetrazole. The β-cyanoethyl phosphite linkage is converted to thephosphorothioate with a sulfurization reagent, such as tetraethylthiuramdisulfide (TETD), 3H-1,2-benzotdithiole-3-one-1,1,-dioxide (Beacaugereagent), or dibenzoyl tetrasulfide. Capping and release from solidsupport follows normal phosphoramidite chemistry procedures.

Dithioate analogs are prepared similarly to phosphorothioate except thata nucleoside 3′ phosphorothioamidite is used. These synthons aregenerated, for example using a suitably protected nucleoside andtris(pyrrolidino)phosphine with a tetrazole activating agent. Theresulting nucleoside diamidite is converted to the phosphorthioamiditewith addition of 2,4 dichlorobenzylmercaptan (see e.g. Caruthers, M. H.,et al. (1992) Chemical Synthesis of Deoxyoligonucleotides andDeoxyoligonucleotide analogs, Meth. Enzy. 211: 3-20). Reaction withdeprotected, solid support bound nucleoside results in a nucleobasepolymer with a thiophosphite triester linkage having the 2,4dichlorobenzylmercaptan. Introduction of elemental sulfur yields thephosphorodithioate derivative. Deprotection is accomplished withthiophenol, which removes the 2,4 dichlorobenzylmercaptan, whiletreatment with base releases the oligonucleotide from the solid support.

Another nucleobase polymer analog embodied in the invention includenucleobase polymers with phosphate triester linkages. Synthesis ofphosphate triester analogs use suitably protected (for example, benzoyl,isobutyryl, or isopropoxyacetyl protecting groups) O-alkyl-N,Ndiisopropylphosphoramidites. Alkyl groups include, but are not limitedto, methyl, ethyl, trifluoroethyl, isopropyl, and neopentyl. In theseembodiments, the chemical groups attached to the free ester aregenerally limited to those groups that are stable to all steps ofconventional phosphoramidite chemistry, including deprotection steps andespecially the procedures and conditions required for deprotection ofprotected amines, such that the resulting linkage is an unchargedphophotriester. Therefore, when such groups are alkyl, the group is, incertain embodiments, an alkyl other than methyl, for example, C₂-C₆linear alkyl, since methyl phophotriesters tend to be less stable thanhigher-order alkyl phosphate triesters. Synthesis follows generalphosphoramidite chemistry with variations in linkage to solid supports(e.g. oxalyl linker) and release from solid support following synthesis(e.g. 25% aq NH₃). Other nucleobase polymer analogs embodied in theinvention include boranophosphates, and phosphofluoridate linkages.Methods for their synthesis are described in Protocols forOligonucleotides and Analogs, Meth. Mol. Biol., Vol 20, Chapters 11 and12, S. Agrawal, Ed., 1993, Humana Press, Inc., which is herebyincorporated by reference.

The sequence-specific nucleobase polymers embodied in the invention arenot limited those with homogeneous internucleotide linkages;sequence-specific nucleobase polymers comprising more than one type ofinternucleotide linkage are also encompassed by the present invention.Thus, mobility-modified sequence-specific nucleobase polymers withcombinations of phosphate diester, phosphate triester, phosphorothioate,and alkylphosphonate internucleoside linkages are all encompassed withinthe scope of the invention. Synthesis of sequence-specific nucleobasepolymers having various combinations of internucleoside linkages arewell know in the art, and references describing their synthesis areincorporated by reference herein (see e.g. Zhou, L. (1994), Synthesis ofphosphorothioate-methylphosphonate oligonucleotide co-polymers, NucleicAcids Res. 22:453-456; Miller, P. S. (1999), Psoralen conjugated triplexforming oligodeoxyribonucleotide containing alternatingmethylphosphonate-phosphate diester linkages: synthesis and interactionswith DNA, Bioconjugate Chem. 10:572-577).

In another aspect, the mobility-modified sequence-specific nucleobasepolymers of the present invention comprise modified bases, a plethora ofwhich have been described in the literature. Methods for synthesizingprotected, modified bases and their incorporation into nucleobasepolymers are well known in the art (see e.g. Connolly, B. A.,Oligodexoynucleotides containing modified basis, In OligonucleotideAnalogs: A practical approach, supra). Base analogs incorporable intonucleic acids, include among others, deoxynapthosine, etheno adenosineand cytidine, 6-thioguanosine, 4-thiothymidine, 7-deaza purines,N⁶-methyl adenosine, O⁶-methyl guanosine, and 2-aminopurine.

In another embodiment of the present invention, the linkages used toattach the mobility-modifying polymer chain to the sequence-specificnucleobase polymer comprise various phosphoester analogs, other thanphosphate diester linkages, including, but not limited to phosphatetriester, alkylphosphonate, and phosphorothiate groups, or othersuitable linkages. As set forth below, synthetic methods to generatevarious linkages can adopt synthetic strategies used in nucleobasepolymer synthesis.

In one aspect, the linkage between the sequence-specific nucleobasepolymer and the mobility-modifying polymer chain is an alkylphosphonatelinkage. In one synthetic scheme, methyldichlorophosphine is reactedwith diisopropylamine to formmethylcholoro-N,N-diisopropylaminophosphine. Addition of suitablyprotected polymer, such as DMT pentaethylene oxide, in the presence ofdiisopropylamine generates methylphosophonamidite derivative with theattached polymer. Coupling of methylphosphoamidite derivative to a freehydroxyl group of a protected, solid support bound oligonucleotideoccurs in the presence of activating agent, such as tetrazole, resultingin a methylphosphonite linkage. Subsequent oxidation, for example withiodine, results in conversion of the methylphosphonite to themethylphosphonate. Other phosphonate linking groups, such asbenzylphosphonate, may be synthesized according to similar chemistryused to generate benzylphosphonate oligonucleotides.

In one embodiment, linking of the mobility-modifying polymer to thesequence-specific nucleobase polymer is accomplished through a phosphatetriester linkage. Various routes of synthesis are available to formphosphate triester linkages between the mobility-modifying polymer andsequence-specific nucleobase polymer. In one embodiment, attaching thepolymer chain to the nucleobase polymer follows phosphoramiditechemistry, as described in detail in Examples 1 and 2 and as illustratedin Schemes (II) and (III), below with ethylene oxide mobility-modifyingpolymers.

In Schemes (II) and (III), DMT represents dimethoxytrityl, iPrrepresents isopropyl and R² and b are as previously defined forstructural formula (I).

Referring to Scheme (II), the R²-N,N-diisopropylaminophosphite 7 isreacted with the free 5′ hydroxyl of a nascent nucleobase polymer 8,thereby generating a nucleobase polymer with a mobility-modifyingpolymer attached to the 5′-terminus by an R²-phosphite triester linkage.Subsequent oxidation with iodine and deprotection with base converts thephosphite linkage to the R²-phosphate triester linkage. The resultantmobility-modified sequence-specific nucleobase polymer 10 is cleaved offthe solid support with base (e.g., NH₄OH, 55° C., 4 hr) to yield 11.Oligonucleotide 11 maybe purified, for example by reverse-phase HPLC,and the remaining trityl group removed with weak acid (e.g., 3%dichloroacetic acid in CH₂Cl₂) to yield mobility-modified nucleobasepolymer 12, which may be further purified, for example by chromatographyon a PD 10 column. Alternatively, the trityl group may be removed priorto cleavage from the solid support and the resultant mobility-modifiednucleobase polymer purified by conventional means.

As will be appreciated by those skilled in the art, when R² is an alkyl,the alkyl phosphoramidite reagent used to couple the mobility-modifyingpolymer segment to the nucleobase polymer may have linear or branchedalkyls of various lengths. Where the phosphate ester is to remainuncharged, the chemical groups attached to the free ester are generallylimited to those groups that are stable to all steps of conventionalphosphoramidite chemistry, including deprotection steps and especiallyto the procedures and conditions required for the deprotection ofprotected amines, such that the resulting linkage is an unchargedphosphate triester. Therefore, when such groups are alkyl, the group isan alkyl other than methyl, for example, C₂-C₆ linear alkyl, since monomethyl phosphate triesters tend to be less stable than higher-order monoalkyl phosphate triesters. The embodiments of the invention alsoenvision attachment of various modified alkyls having functionalmoieties, such as reporters, and ligand molecules. For example,β-cyanoethoxy phosphoramidites with conjugated functional moieties,including but not limited to biotin, psoralen, acridine dye,cholesterol, fluoresceine, rhodamine, 2,4 dinitrophenol,tris-(2,2′-bipyridine rutheium (II) chelate (TBR), and histidines arewell known in the art (see e.g. Smith, T. H. (1999), BifunctionalPhosphoramidite Reagents for the Introduction of Histidyl and DihistidylResidues into Oligonucleotides, Bioconjugate Chem., 10: 647-652; Kenten,J. H. (1992), Improved electrochemiluminescent label for DNA probeassays: rapid quantitative assays of HIV-1 polymerase chain reactionproducts, Clin. Chem, 38:873-9). In general, these functional moietiesare coupled to the phosphoramidite by a hydroxyl group present onpolyoxide or aliphatic spacer arms conjugated to the derivatized moiety.Accordingly, the cognate alkyl phosphoramidite reagent having aderivatized, functional moiety and an attached mobility-modifyingpolymer can be readily synthesized by those skilled in the art byadapting the synthetic scheme provided in the instant application.

In some embodiments of the invention, multiple mobility-modifyingpolymers are attached to the nucleobase polymer. In one embodiment, themultiple mobility-modifying polymers are attached linearly to oneanother, either by way of uncharged linkages such as phosphate triesterlinkages (exemplified by the compounds of structural formula (II)), orby way of charged linkages such as negatively charged phosphate esterlinkages (exemplified by the compounds of structural formula (III)). Amethod for synthesizing these types of mobility-modified nucleobasepolymer polymers using standard phosphoramidite DNA chemistry isillustrated in Scheme (III), below.

In Scheme (III), the various abbreviations and substituents are asdefined for Schemes (I) and (II).

Referring to Scheme (III), support-bound nascent mobility-modifiednucleobase polymer 10, which is synthesized as illustrated in Scheme(II), is treated with a weak acid, in this case 3% dichloroacetic acidin dichloromethane, to remove the DMT protecting group, yieldingsupport-bound compound 13. Support-bound compound 13 is coupled withDMT-protected polyethylene oxide β-cyanoethyl phosphoramidite reagent 14in the presence of an activator such as tetrazole (typically 0.5 M inacetonitrile). Phosphoramidite reagent 14 maybe prepared using standardsyntheses. For example, phosphoramidite reagent 14 may be preparedaccording to Scheme (I) by substituting 2-cyanoethan-1-ol for compound4. Oxidation, such as by reaction with a solution of iodine intetrahydrofuran, 2,6-lutidine and water, yields compound 15. Cleavagefrom the resin, removal of any groups protecting the nucleobases andremoval of the DMT protecting group yields mobility-modified nucleobasepolymer 16. Additional polymers may be added by removing the DMT groupfrom compound 15 and reacting it with phosporamidite reagent 7 or 14.

When the polymers are added sequentially, as for example in an automatedDNA synthesis instrument, addition of additional mobility-modifyingpolymer chains is accomplished by deprotecting the OH protecting groupon the terminus of the mobility-modifying polymer chain and repeatingthe cycles of coupling and oxidation as set forth above. Differentmobility-modifying polymer chains may be added at each cycle to providemobility-modified sequence-specific nucleobase polymers with distinctiveratios of charge to translational frictional drag.

Alternatively, the invention also contemplates mobility-modifiednucleobase polymers in which the polymeric segment is a branched ordendritic structure. An exemplary method for synthesizing aphosphoramidite reagent that can be used in conjunction with standardDNA synthesis chemistry to synthesize such branched and/or dendridicmobility-modified nucleobase polymers is illustrated in Scheme (IV),below. In Scheme (IV), the various abbreviations and substituents are asdefined for Schemes (I) and

Scheme (IV) is similar to Scheme (I), except that a suitably protectedpolymer, such as DMT protected poly(alkylene oxide) 6 substitutes foralcohol 4, thereby forming bis(diisopropylamino)phosphite 20 having anattached polymer. Subsequent coupling of the second mobility-modifyingpolymer 6 generates a phosphoramidite reagent 21 having twomobility-modifying polymer chains. Coupling to the free hydroxyl groupof a solid support bound sequence-specific nucleobase polymer andsubsequent oxidation produces a branched mobility-modifiedsequence-specific nucleobase polymer.

Further addition of mobility-modified polymers are possible. Theprotecting group, for example DMT, on the mobility-modified polymerslinked to the sequence-specific nucleobase polymer are removed with aweak acid, thus generating free hydroxyl groups. Reactions withderivatized N,N-diisopropylaminophosphite having an attachedmobility-modifying polymer results in coupling of the additionalmobility-modifying polymer units to the free hydroxyl groups. Couplingof any additional mobility-modifying polymer units may be limited toonly one of the sequence-specific nucleobase polymer linkedmobility-modifying polymers if the other linked mobility-modifyingpolymer has a nonreactive terminus. Alternatively, protecting thehydroxyl group of one nucleobase polymer linked polymer with protectinggroup that is stable to reagents used to remove DMT protecting groups,such as levulinyl, restricts coupling of any additional polymer units tothe polymer protected with DMT (Iwai, S. (1988), 5′-Levulinyl and2′-tetrahydrofuranyl protection for the synthesis ofoligoribonucleotides, Nucleic Acids Res, 16:9443-56). This orthogonalstrategy removes the protecting groups under mutually exclusiveconditions.

The mobility-modifying phosphoramidite reagent of the present inventionis also added to the 3′-end of a nucleobase polymer, according to SchemeV, which is depicted below:

According to Scheme V, the mobility-modifying phosphoramidite reagent ofthe present invention is condensed with the free 5′-hydroxyl of anucleobase monomer residue that is bound to a solid support (indicatedin Scheme V as), rather than the free 5′-hydroxyl of a nucleobasepolymer bound to a solid support, as depicted in Scheme II. The firststep in Scheme V generates a mobility-modified phosphoramidite polymerbound to the solid support via a nucleobase monomer linker (24). Mildacid treatment of 24 cleaves the DMT protecting group, thereby providinga free hydroxyl moiety upon which a nucleobase polymer is assembled viarepeated cycles of condensation of activated, protected phosphoramiditenucleobase monomers using standard phosphoramidite chemistry, generallyusing an automated instrument. For example, eight further cycles arecarried out, in which the following monomers are added, in order, A, T,G, C, A, T, G, and C.

After addition of the desired specific nucleobase polymer sequence tothe surface-bound mobility-modifying polymer of the present invention,the mobility-modified sequence specific nucleobase polymer, in which themobility-modifying polymer segment is carried at the 3′-end of thenucleobase polymer, is deprotected and cleaved from the solid support,yielding, continuing with the above illustrative synthesis, the productaccording to (29):

Alternatively, the substrate-bound molecule having a mobility-modifyingpolymer attached to the 3′-end of a nucleobase polymer, e.g. structure(28) of Scheme V, is subjected only to a mild acid treatment, removingthe 5′-DMT moiety, and providing a free hydroxyl group on asubstrate-bound nucleobase polymer structure, which would be analogouswith (8) of Scheme II. Subsequent condensation with a mobility-modifyingphosphoramidite reagent of the present invention would provide asequence-specific nucleobase polymer having mobility-modifying polymersegments attached at both the 3′-end and the 5′-end.

another embodiment, a mobility-modified nucleobase polymer issynthesized that comprises a mobility-modifying polymer attached to the5′-end of a first nucleobase polymer as well as to the 3′-end of asecond nucleobase polymer; that is the mobility-modifying polymer is“inserted” within a nucleobase polymer. In one non-limiting illustrativeapproach, a nucleobase polymer comprising a mobility-modifying polymeris synthesized according to Scheme II to provide intermediate (10),which comprises a first nucleobase polymer attached to a solid substrateand carrying a mobility-modifying polymer on the 5′-end of the firstnucleobase polymer. Treatment of (10) with mild acid (3% DCA) to removethe DMT protecting group provides a structure, analogous to compound(25), onto which a second nucleobase polymer is added according toScheme V, thereby providing a mobility-modified nucleobase polymer thatcomprises a first and a second nucleobase polymer in which amobility-modifying polymer is attached to the 5′-end of the firstnucleobase polymer as well as to the 3′-end of the second nucleobasepolymer.

Therefore, the present invention also relates to a mobility-modifiedsequence-specific nucleobase polymer comprising a mobility-modifyingpolymer linked to the 3′-end of a first sequence-specific nucleobasepolymer and to the 5′-end of a second sequence-specific nucleobasepolymer according to Structural formula (IV):

or a salt thereof, wherein:

each R¹¹ is independently selected from the group consisting ofhydrogen, alkyl comprising at least two carbon atoms, aryl, (R⁸)₃Si—where each R⁸ is independently selected from the group consisting oflinear and branched chain alkyl and aryl, base-stable protecting groups,R⁵—X—[(CH₂)_(a)—O]_(b)—(CH₂)_(a)—, protecting group, reporter molecule,and ligand, with the proviso that at least one R¹¹ is not hydrogen;

each X is independently selected from the group consisting of O, S, NHand NH—C(O);

each a is independently an integer from 1 to 6;

each b is independently an integer from 0 to 40;

d is an integer from 1 to 200;

OLIGO¹ is a first sequence-specific nucleobase polymer; and

OLIGO² is a second sequence-specific nucleobase polymer.

OLIGO¹ and OLIGO² are sequence-specific nucleobase polymers, typicallycomprising at least 5 nucleobases. Moreover, where desired,mobility-modifying polymers of the present invention are also added tothe 5′-end of the first nucleobase polymer and/or to the 3′-end of thesecond nucleobase polymer generally according to Schemes V and II,respectively, in addition to the mobility-modifying polymer positionedbetween the first and second nucleobase polymers, according toStructural formula V.

In yet another embodiment, the multiple mobility-modifying polymerchains may be synthesized independently and then attached in a singlestep to the sequence-specific nucleobase polymer. Various schemes may bedevised for synthesizing the multiple mobility-modifying polymer chainsindependently of the sequence-specific nucleobase polymer. In onemethod, a mobility-modifying polymer chain such as pentaethylene oxideis protected with two different protecting groups, which are removableunder mutually exclusive deprotecting conditions. One such orthogonalstrategy is to protect the polymer with dimethoxytrityl at the hydroxylgroup at one terminus and with levulinyl at the hydroxyl group of theother terminus. Alternatively, the polymer is attached to a solidsupport, analogous to attachment of nucleosides to solid supports forsequence-specific nucleobase polymer synthesis, through a bond that isresistant to conditions of DMT deprotection. In either case, treatmentwith weak acid preferentially removes the DMT protecting group, therebyexposing a free OH, while the other hydroxyl group remains protected. Inthe presence of an activating agent, such as tetrazole, a derivatizedphosphoramidite reagent having an attached mobility-modifying polymerreacts with the hydroxyl group, resulting in coupling of the twopolymers. Subsequent oxidation of the intermediate phosphite generatesthe β-cyanoethyl phosphate or phosphate triester linkage depending onthe type of phosphoramidite reagent used for the coupling. Repeating thecycles of deprotection, coupling and oxidation generates linkedmobility-modifying polymer chains, wherein the multiplemobility-modifying polymer chain is protected with DMT at one end andthe other end with levulinyl. Deprotection by removal of the levulinywith hydrazine, or cleavage from the solid support bound polymer,exposes a free hydroxyl, which is then available for synthesizing thederivatized phosphoramidite reagent, for example analkyl-N,N-diisopropylaminophosphite having an attached multiplemobility-modifying polymer chain. Coupling of the derivatizedphosphoramidite reagent to a suitably protected nucleobase polymerresults in attachment of the multiple mobility-modifying polymer chainto the sequence specific nucleobase polymer. Subsequent oxidation withiodine followed by treatment with base removes any protecting groups onthe exocylic amines and β-cyanoethyl groups on the phosphates, withconcomitant release of the mobility-modified sequence specific nucleicacid or nucleobase polymer from the solid support. Treatment with weakacid removes any remaining DMT protecting group on themobility-modifying polymer.

4.6 Methods of Use

The present invention further encompasses methods of usingmobility-modified sequence-specific nucleobase polymers, as well ascompositions comprising a plurality of mobility-modifiedsequence-specific nucleobase polymers, wherein each saidmobility-modified nucleobase polymer optionally has a structureindependently selected from the group consisting of Structural formulae(II) and (III), and wherein each mobility-modified nucleobase polymerhas a distinctive ratio of charge to translational frictional drag, todetect and characterize one or more selected nucleotide sequences withinone or more target nucleic acids.

In one aspect, the present invention provides a method of detecting aplurality of sequences within one or more target nucleic acids,comprising contacting a plurality of mobility-modified sequence-specificnucleobase polymers, wherein each mobility-modified nucleobase polymerhas a structure independently selected from the group consisting ofStructural formulae (II) and (III), with one or more target nucleicacids, generally under conditions that distinguish thosemobility-modified sequence-specific nucleobase polymers that hybridizeto the target nucleic acid, and detecting those mobility-modifiedsequence-specific nucleobase polymers which have hybridized to thetarget nucleic acid.

In one aspect of this method, the target nucleic acids are immobilized.In this aspect, the immobilized target nucleic acids are contacted withmobility-modified sequence-specific nucleobase polymer probes, whichfurther comprise a detectable label, under conditions that distinguishthose probes having sufficient homology to hybridize to the targetnucleic acid. Non-hybridized probes are washed away and hybridizedprobes, which are bound to the target nucleic acid immobilized on themembrane, are detected. Alternatively, the non-hybridized probes arewashed away and hybridized probes are recovered as single-strandedproducts after denaturation of the base-paired structure formed betweenthe mobility-modified sequence-specific nucleobase polymer probe and theimmobilized target nucleic acid.

In another aspect of this method, the target nucleic acid, which may beimmobilized, is contacted with a plurality of sequence-specificnucleobase polymer probes whereby two nucleobase polymer probeshybridize to adjacent sequences of the target nucleic acid such that the5′-end of one nucleobase polymer probe, which generally will carry a5′-phosphate moiety, abuts the 3′-end of the second nucleobase polymerprobe, so that the two nucleobase polymer probes can be covalentlyjoined to one another, in certain embodiments, with a DNA chemical orenzymatic ligating activity, to form a ligated product. In this aspectof the method, the ligated product is formed by the joining of twonucleobase polymer probes, at least one of which comprises a detectablelabel and at least one of which is a mobility-modified sequence-specificnucleobase polymer selected from the group consisting of Structuralformulae (II) and (III), such that the ligated product has a distinctiveratio of charge to translational frictional drag. In a further aspect,three or more nucleobase polymer probes are hybridized to adjacentsequences of a target nucleic acid in such a manner that at least threenucleobase polymer probes can be covalently joined to form a ligatedproduct, wherein at least one of the nucleobase polymer probes so joinedcomprises a detectable label, and at one of the nucleobase polymerprobes so joined is a mobility-modified sequence-specific nucleobasepolymer selected from the group consisting of Structural formulae (II)and (III) such that the ligated product has a distinctive ratio ofcharge to translational frictional drag. Generally, the ligated product,which is hybridized to the target nucleic acid, is released bydenaturation, and the single stranded ligated product having adistinctive ratio of charge to translational frictional drag, isdetected and analyzed, to provide information about the selectednucleotide sequence within the target nucleic acid.

This cycle of hybridization, joining, and denaturation, may be repeatedin order to amplify the concentration of the ligated product formed. Inthis instance, the joining is optionally accomplished by means of athermostable ligating enzyme. These reactions are conveniently carriedout in thermal cycling machines with thermally stable ligases. (Barany,F. (1991), Genetic disease detection and DNA amplification using clonedthermostable ligase, Proc. Natl. Acad Sci. USA 88 (1): 189-193; HousbyJ. N. et al. (2000), Optimised Ligation of oligonucleotides by thermalligases: comparison of T. scotoductus and Rhotothermus marinus DNAligases to other thermophilic ligases, Nuc. Acids Res. 28 (3): E10).

Furthermore, additional nucleobase polymers, which together aresufficiently complementary to the ligated product to hybridize theretoand be covalently joined to one another as above, are also included,thereby affording geometric amplification of the ligated product, i. e.,a ligase chain reaction (Wu, D. Y. and Wallace B. (1989), The ligationamplification reaction (LAR)-Amplification of Specific DNA sequencesusing sequential Rounds of Template Dependent Ligation, Genomics4:560-569; Barany, (1991), Proc. Natl. Acad. Sci. USA, 88:189; Barany,(1991), PCR Methods and Applic., 1:5). To suppress unwanted ligation ofblunted ended hybrids formed between complementary pairs of themobility-modified and second oligonucleotides and the second pair ofoligonucleotides, conditions and agents inhibiting blunted endedligation, for example 200 mM NaCl and phosphate, are included in theligation reaction.

The product of such a ligase chain reaction therefore is a doublestranded molecule consisting of two strands, each of which is theproduct of the joining of at least two sequence-specific nucleobasepolymer probes. Accordingly, in yet another aspect of the presentinvention, at least one of the sequence-specific nucleobase polymersincorporated within the ligase chain reaction product comprises adetectable label, and at one of the sequence-specific nucleobasepolymers is a mobility-modified sequence-specific nucleobase polymerselected from the group consisting of Structural formulae (II) and (III)such that the ligase chain reaction product has a distinctive ratio ofcharge to translational frictional drag.

In another aspect of the oligonucleotide ligase assays described above,mismatches, i.e. non-complementary nucleobases, existing betweenselected nucleotide sequences within the target nucleic acid and eitheror both of the mobility-modified sequence-specific nucleobase polymerand the second oligonucleotide interfere with the ligation of the twonucleobase polymers either by preventing hybrid formation or preventingproper joining of the adjacent terminal nucleotide residues. Thus, whenthe binding conditions are chosen to permit hybridization of bothnucleobase polymers despite at least one mismatch, the formation of amobility-modified ligated product reveals the sequence of the selectednucleotide sequence as it exists within the target nucleic acid, atleast with respect to the terminal, adjacent residues of the twonucleobase polymers. Those skilled in the art are well versed inselecting appropriate binding conditions, such as cation concentration,temperature, pH, and oligonucleotide composition to selectivelyhybridize the nucleobase polymers to the selected nucleotide sequenceswithin the target nucleic acid.

Since the base pairing of terminal adjacent residues affects ligation,in one embodiment the nucleobase polymer providing the 3′ terminalnucleobase involved in the joining reaction is designed to be perfectlycomplementary to the target sequence while the nucleobase polymerproviding the 5′ terminal nucleobase residue involved in the joiningreaction is designed to be perfectly complementary in all but the 5′terminal nucleobase. In another embodiment, the nucleobase polymers aredesigned such that the nucleobase polymer providing the 3′ terminalnucleobase is perfectly complementary except for the 3′ terminalnucleobase residue while the oligonucleotide providing the 5′ terminalnucleobase is perfectly complementary (Wu, D. Y. and Wallace, B.,(1989),Specificity of nick-closing activity of bacteriophage T4 DNA ligase,Gene 76: 245-254; Landegren, U. et al. (1988) A ligase mediated genedetection technique, Science 2241: 1077-1080).

In a modification of the method set forth above, the mobility-modifiedsequence-specific nucleobase polymer probe comprises a nucleobasesequence that is complementary to the target sequence, but comprises anon-terminal mismatch with respect to non-target sequences. In thisaspect of the invention, the composition of the mobility-modifiedsequence-specific nucleobase polymer probe and the nature of theexperimental conditions are such that the probe will only hybridize tothe target sequence. In this embodiment for example, a second nucleobasepolymer that hybridizes to the target nucleobase, either upstream ordownstream of the hybridized mobility-modified sequence-specificnucleobase polymer probe, may be ligated to that probe to form theligated, mobility-modified product that is diagnostic of the presence ofthe target nucleotide sequence.

In a further modification of the embodiment set forth above, themobility-modified sequence-specific nucleobase polymer is hybridized toa selected nucleotide sequence within a target nucleic acid that isimmediately adjacent to the site of interest. A second sequence-specificnucleobase polymer is hybridized to the selected region within thetarget nucleic acid such that the hybridized oligonucleotides areseparated by a gap of at least one nucleotide residue. In anotherembodiment, the length of the gap is a single nucleotide residuerepresenting a single polynucleotide polymorphism in the target nucleicacid. Following hybridization, the complex, which consists of the twonucleobase polymers hybridized to the target nucleic acid, is treatedwith a nucleic acid polymerase in the presence of at least onedeoxyribonucleoside triphosphate. If the deoxyribonucleosidetriphosphate(s) provided are complementary to the targetpolynucleotide's nucleotide residues which define the gap, thepolymerase fills the gap between the two hybridized nucleobase polymers.Subsequent treatment with ligase joins the two hybridizedoligonucleotides to form a ligated, mobility-modified product, whichcan, in one embodiment, be separated from the template by thermaldissociation, thereby providing a diagnostic product having adistinctive ratio of charge to translational frictional drag. Thisdiagnostic product will generally comprise a reporter molecule, whichmay be included within either of the ligated nucleobase polymers, beattached to the one or more nucleobases added by the polymerizingactivity, or be added subsequent to the covalent joining of thenucleobase polymers, using methods disclosed infra. By treating withpolymerase in the presence of fewer than four nucleoside triphosphates,the nucleotide residues comprising the gap may be determined. Furtheramplification of ligated mobility-modified product is achieved byrepeated cycles of denaturation, annealing, nucleic acid polymerase gapfilling, and ligation in the presence of at least one of the nucleosidetriphosphates.

If the treatment with nucleic acid polymerase occurs in the presence ofone labeled nucleoside triphosphate or a mixture containing one labeledand 3 unlabeled nucleoside triphosphates, ligated mobility-modifiedproducts comprising at least one incorporated, labeled nucleoside arereadily detected upon electrophoretic separation of the labeledmobility-modified ligated products. Modification of the nucleotidemixture to one having one labeled nucleoside triphosphate and threechain terminating nucleoside triphosphates suppresses unwanted ligationof oligonucleotides with incorrectly incorporated nucleotide residues.

Mobility-modified sequence-specific nucleobase polymers of the presentinvention are also useful as primers for nucleic acid sequence analysisby the chain termination method, a method well known to those skilled inthe art. In one embodiment, mobility-modified nucleobase polymers arehybridized to target nucleic acid and extended by a nucleic acidpolymerase in the presence of a mixture of nucleoside triphosphates anda chain terminating nucleoside triphosphate. The polymerase reactiongenerates a plurality of chain terminated mobility-modified nucleicacids fragments, which are separated, for example by capillaryelectrophoresis. Chain termination by the incorporated chain terminatingnucleoside triphosphate identifies the 3′ terminal residue of theterminated nucleic acid fragment.

For the purposes of detecting the chain terminated species, varioussubstituents of the mobility-modified nucleic acid fragments areamenable to conjugation with detectable reporter molecules. Theseinclude the functional groups on the mobility-modifying polymer, thephosphate triester group linking the mobility-modifying polymer to thesequence-specific nucleobase polymer, or nucleoside triphosphateprecursors, including the chain terminators, incorporated into thenucleic acid. Detectable reporter molecules may be radioactive,chemiluminescent, bioluminescent, fluorescent, or ligand molecules. Inone embodiment, the detectable label is a fluorescent molecule, forexample fluorescein isothiocyanate, Texas red, rhodamine, and cyaninedyes and derivatives thereof. In another embodiment, the fluorescentdyes are mobility-modified to reduce the variations in electrophoreticmobility of nucleic acids caused by the fluorescent label (see e.g. Ju,J. et al. (1995). Design and synthesis of fluorescence energy transferdye-labeled primers and their application for DNA sequencing andanalysis, Anal. Biochem. 231: 131-40; Metzker, et al. (1996)Electrophoretically uniform fluorescent dyes for automated DNAsequencing, Science 271: 1420-22; Hung, S. C. et al. (1997) Comparisonof fluorescence energy transfer primers with different donor-acceptordye combinations, Anal Biochem. 252: 77-88; Tu, O. et al. (1998) Theinfluence of fluorescent dye structure on the electrophoretic mobilityof end-labeled DNA, Nucleic Acids Res. 26: 2797-2802)

In one embodiment, the mobility-modified sequence-specific nucleobasepolymers of the present invention are used within a format forsequencing selected regions within a target polynucleotide wherein oneof four spectrally resolvable fluorescent molecules is used to label thenucleic acid fragments in reactions having one of four chain terminatingnucleoside triphosphates. In another aspect of this embodiment, themobility-modified sequence-specific nucleobase polymers of the presentinvention are used for sequencing selected regions within a targetpolynucleotide wherein one of four spectrally resolvable fluorescentmolecules is used to label an oligonucleotide primers in a reactioncontaining one of four chain terminating nucleoside triphosphates. Thus,in both aspects of this embodiment, detecting the fluorescent color ofthe chain terminated nucleic acid fragment identifies the 3′ terminalnucleotide residue. Separation of the chain-terminated products byelectrophoresis, typically in a single gel lane or capillary, along withsimultaneous on-line detection of four spectrally resolvable fluorescentmolecules allows rapid sequence determination from the colors of theseparated nucleic acid fragments (Prober, J. M. et al. (1985), A Systemfor Rapid DNA Sequencing with Fluorescent Chain TerminatingDideoxynucleotides, Science 238: 336-341; Karger, A. E. et al., (1991),Multiwavelength Fluorescence Detection for DNA Sequencing UsingCapillary Electrophoresis, Nucleic Acids Res. 19 (18):4955-62).

When the nucleotide sequence of interest is a small region of the targetnucleic acid, for example a site including single nucleotidepolymorphism, modified sequencing formats, optionally, are used. In onesuch embodiment, a mobility-modified sequence-specific nucleobasepolymer is hybridized in a sequence-specific manner such that the3′-terminal nucleotide residue of the mobility-modifiedsequence-specific nucleobase polymer is immediately adjacent to the siteof interest. The hybridized nucleobase polymer is extended by a nucleicacid polymerase in the presence of at least one chain terminatingnucleoside triphosphate extends the oligonucleotide by one nucleotide ifthe chain terminating nucleotide is complementary to the target nucleicresidue immediately downstream of the 3′-terminus of the hybridizedmobility-modified sequence-specific nucleobase polymer. Separation anddetection of the extended, mobility-modified sequence-specificmobility-modified sequence-specific nucleobase polymer provides theidentity of the residue immediately adjacent to the hybridizedmobility-modified sequence-specific nucleobase polymer primer. In thisembodiment, the use of a plurality of different mobility-modifiedsequence-specific nucleobase polymers permits the simultaneous detectionand analysis of a plurality of target sequences in a single separation.

Detecting the extended primer is accomplished by including a reportermolecule conjugated to the extended, mobility-modified sequence-specificnucleobase polymer are used as primers in the same manner as describedabove for standard sequencing reactions. Thus, in one embodiment, thechain terminating nucleoside triphosphate is labeled with one of fourspectrally resolvable fluorescent molecules such that the fluorescentlabel uniquely identifies the chain terminating nucleotide. Thecomposition of the residue immediately adjacent to the hybridizedoligonucleotide primer is then readily ascertained from the colors ofthe extended oligonucleotide primer. As will be apparent to thoseskilled in the art, this modified sequencing format is adaptable toother mixtures of fluorescently labeled chain terminating nucleosidetriphosphates. Thus the embodiments encompass nucleotide combinationshaving two or four chain terminating nucleoside triphosphates whereinonly one chain terminator is labeled with one of four resolvablereporter labels. Mixing the products of the extension reactions,followed by separation and detection of the extended products in asingle gel lane or capillary provides the ability to determine allpossible sequence variations at the nucleotide residue adjacent to thehybridized primer. Further increase in sensitivity of the methods arepossible by using substantially exonuclease-resistant chain terminators,such as those which form thio-ester internucleotide linkages, to reduceremoval of incorporated chain terminators by polymerase associatedexonuclease.

In another embodiment, mobility-modified sequence-specific nucleobasepolymers are used in polymerase chain reactions (PCR) to detect andamplify selected nucleotides within one or more target nucleic acids(Mullis, K., U.S. Pat. No. 4,683,202; Saiki, R. K., et al., EnzymaticAmplification of β-Globin Genomic Sequences and Restriction SiteAnalysis for Diagnosis of Sickle Cell Anemia, In PCR: A practicalapproach, M. J. McPherson, P. Quirke, and G. R. Taylor, Eds., OxfordUniversity Press, 1991). In this aspect of the present invention, thedetection method involves PCR amplification of nucleotide sequenceswithin the target nucleic acid. In this aspect, a target nucleic acid,which may be immobilized, is contacted with a plurality ofsequence-specific nucleobase polymers, two of which hybridize tocomplementary strands, and at opposite ends, of a nucleotide sequencewithin the target nucleic acid. Repeated cycles of extension of thehybridized sequence-specific oligonucleotides, optionally by athermo-tolerant polymerase, thermal denaturation and dissociation of theextended product, and annealing, provide a geometric expansion of theregion bracketed by the two nucleobase polymers. The product of such apolymerase chain reaction therefore is a double-stranded moleculeconsisting of two strands, each of which comprises a sequence-specificnucleobase polymer probe. In this aspect of the present invention, atleast one of the sequence-specific oligonucleotides is amobility-modified sequence-specific nucleobase polymer selected from thegroup consisting of Structural formulae (II) and (III) such that thedouble stranded polymerase chain reaction product has a distinctiveratio of charge to translational frictional drag. The polymerase chainreaction product formed in this aspect of the invention furthercomprises a label, which may be incorporated within either of thesequence-specific nucleobase polymer probes used as primers, or it maybe incorporated within the substrate deoxyribonucleoside triphosphatesused by the polymerizing enzyme. In yet another aspect, the polymerasechain reaction product formed is analyzed under denaturing conditions,providing separated single stranded products. In this aspect, at leastone of the single stranded products comprises both a label and amobility-modified sequence-specific nucleobase polymer primer selectedfrom the group consisting of Structural formulae (II) and (III) suchthat the single-stranded product derived from double stranded polymerasechain reaction product has a distinctive ratio of charge totranslational frictional drag. As is well known in the art, such asingle-stranded product may also be generated by carrying out the PCRreaction with limiting amounts of one of the two sequence-specificnucleobase polymer probes used as a primer. By using distinctivemobility-modified sequence-specific nucleic acids or nucleobase polymersas primers, the PCR reaction can detect many selected regions within oneor more target polynucleotides in a single assay by allowing separationof one PCR product from another. Moreover, those skilled in the art willrecognize that using various combinations of nucleobase polymer primersprovides additional ways to generate distinctive mobility-modified PCRproducts. For example, a combination of a mobility-modified nucleobasepolymer and a second nucleobase polymer primer pair in the PCR reactiongenerates a PCR product with a single mobility-modified strand. On theother hand, a combination of a mobility-modified nucleobase polymer anda second nucleobase polymer, which is also mobility-modified, generatesa PCR product having both strands that are mobility-modified, thusdistinguishing itself from the PCR product with one mobility-modifiedstrand. Thus, by varying the type of mobility-modifying group and thenucleic acid strands that are mobility-modified, the embodiments enlargethe capacity to detect multiple target segments.

Detection of the PCR products may be accomplished either duringelectrophoretic separation or after an electrophoretic separation.Intercalating dyes such as ethidium bromide, ethidium bromide dimers,SYBR® Green, or cyanine dye dimers such as TOTO, YOYO and BOBO areavailable for post separation detection (Haugland, R. P. Handbook ofFluorescent Probes and Research Chemicals, 6^(th) ed, Molecular Probes,Inc., 1996). Alternatively, the PCR products further comprise reportermolecules, including but not limited to radioactive, chemiluminescent,bioluminescent, fluorescent, or ligand molecules that permit detectioneither during or subsequent to an electrophoretic separation. Methodsfor labeling the mobility-modified PCR products follow the generalschemes presented for labeling in other methods described infra.

Detecting a selected nucleotide sequence within a target nucleic acid byPCR amplification also encompasses identifying sequence variationswithin segments of the target nucleic acid. These variations include,among others, single nucleotide polymorphisms and polymorphisms invariable nucleotide tandem repeats (VNTR) and short tandem repeats(STR), such as those defined by sequence tag sites (STS). Identifyingpolymorphic loci are of particular interest because they are oftengenetic markers for disease susceptibility (see e.g. Gastier, J. M.,(1995), Hum Mol Genet, 4(10):1829-36; Kimpton, C. P., (1993), AutomatedDNA profiling employing multiplex amplification of short tandem repeatloci, PCR Methods Appl., 3(1): 13-22). If the polymorphisms relate tovariations in VNTR or STR sequences, direct analysis of PCR productswithout further treatment suffices for detecting polymorphisms since theproducts differ in nucleotide length. The presence of mobility-modifiedPCR products, however, expands the capability of the PCR analysis todetect multiple polymorphic loci in a single reaction.

If the polymorphisms relate to single nucleotide differences, thevariations are detectable by conducting PCR reactions using nucleobasepolymer primers designed to have mismatches with the selected nucleicacid sequence within a target nucleic acid. The presence of intentionalmismatches within the duplex formed by hybridization of the nucleobasepolymer primer and the selected nucleic acid sequence within the targetnucleic acid affects the thermal stability of those duplex molecules,which is reflected in the T_(m) of those structures and thus, underselected conditions, results in preferential amplification of one targetsegment as compared to another. Such allele-specific polymerase chainreactions permit identification of mutations in single cells, or tissuescontaining a low copy number of one selected nucleotide sequencesamongst a high background of other nucleotide seqeunces within one ormore target nucleic acids (Cha, R. S., (1993), Mismatch amplificationmutation assay (MAMA): application to the c-H-ras gene., PCR MethodsAppl., 2(1) 14-20; Glaab., W. E. et al., (1999), A novel assay forallelic discrimination that combines fluorogenic 5′ nuclease polymerasechain (TaqMan®) and mismatch amplification mutation., Mutat. Res. 430:1-12).

In yet another aspect, single nucleotide differences are distinguishedthrough analysis of higher order conformations of single strandednucleic acids that form in a sequence dependent manner. In thisembodiment, single stranded nucleic acids are generated by dissociatingthe PCR products into single strands, or by preferentially amplifyingone strand by using limiting amounts of one primer in the PCR reaction(i.e. single-sided PCR). Under selected conditions, the single strandednucleic acids are allowed to form higher order structures byintramolecular hydrogen bonding of the single stranded nucleic acid.Those skilled in the art are well versed in defining such permissiveconditions (i.e. temperature, denaturant concentration, pH, cationconcentration etc.) for forming the higher order structures. Theseconformations, which are sequence dependent and which therefore can beextremely sensitive to single nucleotide changes, affect theelectrophoretic mobility of the nucleic acid, and thus reveal variationin a selected nucleotide sequence within a target nucleic acid by theirunique electrophoretic mobility profiles. To enhance formation of higherorder structures modifications are introduced into the nucleobasepolymer primers used for the PCR reactions. For example, a nucleobasepolymer primer is engineered with additional bases complementary to apart of the selected nucleotide sequence within a target nucleic acidcontaining the sequence variation, such that higher order conformationsform when the additional bases on the nucleobase polymer primer“snapback” or re-anneal to the normal sequence but not to variantsequences (Wilton, S. D. (1998), Snapback SSCP analysis: engineeredconformation changes for the rapid typing of known mutations, Hum.Mutat. 11(3): 252-8). Since the reliability of detecting singlenucleotide variations is affected by size of the single strandednucleobase polymer, conformation analysis using mobility-modifiedsequence-specific nucleobase polymers, with each having a distinctiveratio of charge to translational frictional drag, permits detection of aplurality of selected nucleotide sequences within a target nucleic acidwhile maintaining the optimal length needed for forming higher orderstructures (Sheffield, V. C., (1993), The sensitivity of single strandedconformation polymorphism analysis for the detection of single basesubstitutions, Genomics 16 (2): 325-32).

In yet another aspect of the invention, mobility-modifiedsequence-specific nucleobase polymers are cleaved to detect selectednucleotide sequences within one or more target nucleic acids. In onesuch embodiment, mobility-modified sequence-specific nucleobase polymersare hybridized to selected nucleotide sequences within one or moretarget nucleic acids. In another embodiment, mobility-modified PCRproducts comprising at least one mobility-modified sequence-specificnucleobase polymer serve as substrates for sequence-specific enzymes,such as restriction enzymes. Digestion of the substrates by the enzymescreates cleaved products having a distinctive ratio of charge totranslational frictional drag, which provides information about sequencecomposition of the target polynucleotides. This form of restrictionfragment length polymorphism (RFLP) analysis is well known to thoseskilled in the art (see e.g., Kidd, I. M., (1998), A multiplex PCR assayfor the simultaneous detection of human herpesvirus 6 and humanherpesvirus 7, with typing of HHV-6 by enzyme cleavage of PCR products,J. Virol. Methods 70 (1): 29-36; Gelernter, J., (1991), Sequence taggedsites (STS) Taq I RFLP at dopamine beta-hydroxylase, Nucleic Acids. Res.19 (8): 1957).

In another aspect, mobility-modified nucleobase polymers hybridized toselected nucleotide sequences within a target nucleic acid, whereinthere is at least one nucleobase not complementary to the correspondingnucleobase in the target nucleic acid, are treated with agents thatspecifically cleave the non-base-paired nucleotide residues. Generally,the unpaired residue occurs on the hybridized mobility-modifiednucleobase polymer (Bhattacharya, et al., (1989), Nucleic Acids. Res.17, 6821-6840). Although chromosomal DNA may serve as the target nucleicacids, target nucleic acids are cloned DNA fragments comprising selectednucleotide sequences of a target nucleic acid, or PCR amplificationproducts comprising selected nucleotide sequences of a target nucleicacid.

Cleavage may be accomplished with either chemical or enzymatic reagents.In chemical cleavage reactions, the hybrids containing at least onenon-complementary nucleobase, are treated with chemicals whichspecifically modify the unpaired residue, rendering the internucleotidelinkage of the modified nucleoside susceptible to hydrolysis. Suitablechemical agents include but are not limited to carbodiimide, osmiumtetraoxide, hydroxylamine or potassium permanganate/tetraethylammoniumchloride (Ellis, T. P., et al., (1998), Chemical cleavage of mismatch: anew look at an established method, Hum Mutat. 11: 345-53; Roberts, E.,(1997), Potassium permanganate and tetraethylammonium chloride are safeand effective substitute for osmium tetraoxide in solid phasefluorescent chemical cleavage mismatch, Nucleic Acids. Res. 25:3377-78). The use of potassium permanganate/tetraethylammonium chloriderather than osmium tetraoxide enhances cleavage at T/G mismatched pairs.

Enzymatic cleaving reagents encompass a variety of nucleases whichrecognize unpaired regions. These include but are not limited to singlestranded specific nucleases such as S1 nuclease from Aspergillus oryzue,P1 from Penicillum citrinum, and mung bean nuclease (Shenk, et al.,(1975) Proc. Natl. Acad. Sci. USA 72 989-93). Although these nucleasesare less reactive towards single nucleotide mismatches, they can digestunpaired residues created by longer insertions and deletions (Dodgson,J. B. et al., (1977), Action of single-stranded specific nucleases onmodel DNA heteroduplexes of defined size and sequence, Biochemistry,16:2374-49). Cel 1 and SP endonucleases show activity toward unpairednucleotide residues resulting from nucleotides sequence variationscomprising deletions, insertions, and missense mutations, withinselected nucleotide sequences of target nucleic acids. (Oleykowski, C.A., (1998) Mutation detection using a novel plant endonuclease, NucleicAcids. Res. 26: 4597-602; Yeung, A. T.,U.S. Pat. No. 5,869,245).Resolvases from various sources, such as bacteriophage and yeast,represent yet another class of cleaving enzymes useful in thisembodiment of the invention. Representative examples of resolvasesinclude but are not limited to phage encoded T4 endonuclease VII and T7endonuclease I, both of which cleave at mismatches (Cotton, R. G. H.,U.S. Pat. No. 5,958,692; Solaro, et al, (1993), Endonuclease VII ofPhage T4 Triggers Mismatch Correction in vitro. J. Mol. Biol. 230:868-877; (Chang, D. Y. et al., (1991), Base mismatch specificendonuclease activity in extracts from Saccharomyces cerevisiae; NucleicAcids Research 19 (17): 4761-66).

In another aspect, of the present invention encompasses methods thatprevent cleavage at unpaired residues. Proteins, including but notlimited to the MutS protein of E. coli., bind to sites of singlenucleotide mismatches in duplex nucleic acid structures (Su, S. S. etal., (1986), Escherichia coli mutS encoded protein binds to mismatchedDNA base pairs, Proc. Natl Acad. Sci. USA 83: 5057-5061). The MutSprotein is part of the methylation directed E coli. MutH/S/L mismatchrepair system, homologs of which are present in other bacteria, yeastand mammals (Eisen, J. A., (1998), A phylogenetic study of the MutSfamily of proteins, Nucleic Acids. Res. 26: 4291-300; Alani, E. (1996),The Saccharomyces cerevisiae Msh2 and Msh6 proteins form a complex thatspecifically binds to duplex oligonucleotides containing mismatched DNAbase pairs, Mol. Cell Biol. 16: 5604-15; Modrich, P. et al., (1996),Mismatch repair in replication fidelity, genetic recombination andcancer biology, Annu. Rev. Biochem. 65: 101-33). Therefore in oneembodiment of the invention, duplex structures comprising at least onenon-base-paired nucleobase unit formed by hybridization of amobility-modified sequence-specific nucleobase polymer with a selectednucleotide sequence within a target nucleic acid, are treated withmismatch binding proteins such as MutS and then exposed to one or moreexonucleases which degrade the duplex strands in a unidirectionalfashion. A bound mismatch binding protein inhibits further action of theexonuclease on the strand containing the mismatch, thereby providingnucleic acid products of defined length and which possess a distinctiveratio of charge to translational frictional drag (Ellis, L. A., (1994),MutS binding protects heteroduplex DNA from exonuclease digestion invitro: a simple method for detecting mutations, Nucleic Acids Res. 22(13):2710-1; Taylor, G. R., U.S. Pat. No. 5,919,623). Unidirectionalexonucleases suitable for use in this assay include, but are not limitedto exonuclease III, bacteriophage λ exonuclease, and the 3′ to 5′exonucleases of T7 DNA polymerase, T4 DNA polymerase, and Vent® DNApolymerase.

In yet another aspect, a mobility-modified sequence-specific nucleobasepolymer is used in a cleavage based method of detecting selectednucleotide sequences within a target nucleic acid may be a DNA-RNA-DNAnucleobase polymer, where an internal RNA segment is flanked by DNAsegments. This tripartite mobility-modified sequence-specific nucleobasepolymer is hybridized to a selected nucleotide sequence within a targetnucleic acid at a temperature below the T_(m) of the overall, i.e.tripartite nucleobase polymer. Digestion of this duplex structure withan appropriate RNase, hydrolyzes only the RNA portion of the DNA-RNA-DNAnucleobase polymer when hybridized to a DNA template. In one embodiment,the RNase is a thermo-stable RNase H (Bekkaoui, F., (1996), Cyclingprobe technology with RNase H attached to an oligonucleotide,Biotechniques, 20 (2): 240-8). If the temperature of the reactionmaintained above the T_(m) of the flanking DNA segments remaining afterdigestion of the internal RNA segment, those DNA segments dissociate,thus allowing another DNA-RNA-DNA oligomer to associate with the targetpolynucleotide. Repeated hybridization, RNA cleavage, and dissociationof the flanking DNA segments amplifies the level of detectabledissociated DNA segments. The reaction temperature, in one embodiment,is held constant during the amplification process, thus obviating anyneed for thermal cycling (Duck, P. (1990), Probe amplifier system basedon chimeric cycling oligonucleotides, Biotechniques 9: 142-48; Modrusan,Z. (1998) Spermine-mediated improvement of cycling probe reaction, Mol.Cell Probes 12: 107-16). In this aspect of the present invention, thesequence-specific DNA-RNA-DNA nucleobase polymer used comprise at leastone mobility-modifying polymer and at least one reporter moleculeattached to either or both of the flanking DNA segments, therebyproviding a labeled digestion product having a distinctive ratio ofcharge to translational frictional drag.

In another aspect, detection of selected nucleotide sequences within oneor more target nucleic acids based on cleavage of a mobility-modifiednucleobase polymer relies upon cleavage substrates formed by invasivehybridization, as described in Brow et al. U.S. Pat. No. 5,846,717. Inthis embodiment, the 5′-portion of a mobility-modified sequence-specificnucleobase polymer, which comprises a reporter molecule and which ishybridized to a target nucleic acid, is displaced by a second nucleobasepolymer that hybridizes to the same region and thereby exposing thatdisplaced sequence to cleavage with a cleaving reagent. In practicingthe embodiment, the target nucleic acid is contacted with amobility-modified sequence-specific nucleobase polymer and with a secondnucleobase polymer. The mobility-modified sequence-specific nucleobasepolymer has a 5′-segment complementary to a second region of theselected nucleotide sequence contained within a target nucleic acid anda 3′-portion complementary to a third region of the selected nucleotidesequence contained within a target nucleic acid, wherein the secondregion is downstream from the third region. The second nucleobasepolymer has a 5′-segment complementary to a first region of the selectednucleotide sequence contained within a target nucleic acid and a3′-segment complementary to the second region of the selected nucleotidesequence contained within a target nucleic acid, wherein the firstregion is downstream from the second region. Under selected conditions,hybrids form in which the mobility-modified sequenced specificnucleobase polymer and the second nucleobase polymer hybridize to thetarget polynucleotide such that the second nucleobase polymer displacesthe 5′ portion of the hybridized mobility-modified sequence-specificnucleobase polymer, whereas the 3′ portion of the mobility-modifiedsequence-specific nucleobase polymer and the 5′ portion of the secondnucleobase polymer remain annealed to the selected nucleotide sequencecontained within a target nucleic acid. The displaced strand, which is asingle stranded segment that is not base-paired corresponds to the5′-end of the mobility-modified sequence-specific nucleobase polymerthen serves as a substrate for cleavage nucleases, thus producingdiscrete mobility-modified digestion products having distinct ratios ofcharge to translational frictional drag that reflect presence ofspecific sequences on the target polynucleotide.

Cleaving enzymes recognizing displaced strands are either naturallyoccurring nucleases or modified nucleases. Naturally occurringstructure-specific nucleases include, but are not limited to Pyrococcuswoesii FEN-1 endonuclease, thermostable Methoanococcus jannaschii FEN-1endonucleases, yeast Rad2, and yeast Rad1/Rad10 complex (Kaiser et al.,U.S. Pat. No. 6,090,606, Cleavage Reagents; Kaiser, et al. U.S. Pat. No.5,843,669, Cleavage of nucleic acid using thermostable Methoanococcusjannaschii FEN-1 endonucleases). Other structure-specific enzymessuitable for the cleaving reaction are those derived from modificationsof known nucleases and polymerases (Dahlberg et al., U.S. Pat. No.5795763, Synthesis Deficient Thermostable DNA Polymerase; Dahlberg etal., U.S. Pat. No. 6,614,402, 5′ Nucleases Derived from Thermostable DNAPolymerase). Modified polymerase that lack polymerase activity but stillretain 5′-nuclease activity, are also used as cleaving reagents.

Another embodiment is directed toward the use of the mobility-modifyfingpolymers of the present invention in “invader assays,” which areSNP-identifying procedures based upon flap endonuclease cleavage ofstructures formed by two overlapping nucleobase polymers that hybridizeto a target nucleic acid (see e.g. Cooksey et al., 2000, AntimicrobialAgents and Chemotherapy 44: 1296-1301). Such cleavage reactions releaseproducts corresponding to the 5′-terminal nucleobase(s) of the“downstream” nucleobase polymer. Where those cleavage products arelabeled and can be separated from the uncleaved nucleobase polymer, aninvader assay can be used to discriminate single base differences in,for example, genomic sequences or PCR-amplified genomic sequences.

Attachment of the mobility-modifying polymers of the present inventionto the labeled 5′-terminus of the downstream nucleobase polymer used inan invader assay provides detectably-labeled cleavage products withdistinctive charge to translational frictional drag ratios. Accordingly,a plurality of SNP's are analyzed simultaneously using a plurality ofsequence-specific downstream nucleobase polymers, wherein thesequence-specific downstream nucleobase polymers comprise amobility-modifying polymer of the present invention attached to thelabeled 5′-terminus, such that the labeled product generated by flapendonuclease cleavage at each SNP has a distinctive charge totranslational frictional drag ratio.

In a further aspect of the invader assay, for example, the downstreamnucleobase polymer, which carries a label and a first mobility-modifyingpolymer of the present invention attached to the 5′-terminus, furthercomprises a second mobility-modifying polymer attached to the3′-terminus. The presence of the second mobility-modifying polymerincreases the sensitivity of the invader assay by enhancing thedifference between the electrophoretic mobility of the flap endonucleasegenerated product, comprising the 5′-terminus, label, and firstmobility-modifying polymer, and the electrophoretic mobility of theuncleaved downstream nucleobase polymer. Accordingly, the secondmobility-modifying polymer has a molecular weight of at least 2000. Inother embodiments, the second mobility-modifying polymer has a molecularweight of at least 5,000, at least 10,000, at least 20,000, and at least100,000. In one embodiment, the second mobility-modifying polymer is amobility-modifying polymer of the present invention, while in otherembodiments, the second mobility-modifying polymer is amobility-modifying polymer of the art, which is, in one illustrative,non-limiting example, an uncharged mono methyl polyethyleneglycolpolymer. Moreover, the second mobility-modifying polymer may comprise amixture of species of different molecular weight, provided that thosespecies do not interfere substantially with detection of the signalproduct, i.e., the flap endonuclease generated product, comprising the5′-terminus, label, and first mobility-modifying polymer (see Example 5,below).

More generally, in other embodiments of the present invention, invaderassays are performed in which the downstream oligonucleobase polymercomprises a label and a mobility-modifying polymer of the presentinvention attached to a first region of the downstream oligonucleobasepolymer, and a second, high-molecular weight mobility-modifying polymerattached to a second region of the downstream oligonucleobase polymer,wherein first and second regions are separated by the flap endonucleasecleavage site. One aspect of this embodiment is described above and inExample 5, wherein the label and mobility-modifying polymer of thepresent invention are attached to the 5′-end of the sequence-specificoligonucleobase polymer and a second, high molecular weightmobility-modifying polymer is attached to the 3′-end of thesequence-specific oligonucleobase polymer. In other embodiments, forexample, a second, high molecular weight mobility-modifying polymer isattached, via a linker arm nucleotide residue, to the sequence-specificnucleobase polymer, rather than at the 5′-end or 3′-end of thesequence-specific nucleobase polymer. Accordingly, the second, highmolecular weight mobility-modifying polymer, is attached at anynucleobase residue within the second region of the downstream nucleobasepolymer, or to the 5′-end or 3′-end, whichever is included within thesecond region of the downstream oligonucleobase polymer. Similarly, insome embodiments, the label, which is a fluorescent dye in certainnon-limiting examples, is also attached via a linker arm nucleotideresidue at any nucleobase residue within the first region of thedownstream nucleobase polymer. Synthesis of such linker arm nucleotidesand the coupling of, inter alia, a fluorescent dye or an uncharged monomethyl polyethyleneglycol polymer to the linker, are within the scope ofthe art (see e.g., Section 4.5 above). Moreover, e.g., linker armnucleoside phosphoramidite monomers, as well as linker arm nucleosidephosphoramidite monomers comprising flourescent moieties, arecommercially available (Glen Research, Inc., Sterling, Va.). In theseembodiments, the mobility-modifying polymer of the present invention isattached to the first region of the downstream nucleobase polymer, wherethe point of attachment may be at the 5′-end or the 3′-end, whichever isencompassed within the first region of the downstream nucleobasepolymer, or the mobility-modifying polymer of the present invention maybe incorporated within the first region of the downstream nucleobasepolymer, providing a molecule according to Structural formula (IV).Therefore, in each of these embodiments, the presence of the second highmolecular weight mobility-modifying polymer attached to the secondregion of the downstream nucleobase polymer increases the sensitivity ofthe invader assay by enhancing the difference between theelectrophoretic mobility of the flap endonuclease generated productcomprising a label and a mobility-modifying polymer of the presentinvention, i.e., the first region of the downstream oligonucleobasepolymer, and the electrophoretic mobility of the uncleaved downstreamnucleobase polymer.

In a still further embodiment of an invader assay, the downstreamnucleobase polymer carries a label and a first mobility-modifyingpolymer, which is in one non-limiting embodiment, a standard PEOmobility-modifying polymer of the art, that is attached to the firstregion of the downstream nucleobase polymer, and a second, highmolecular weight mobility-modifying polymer attached to the secondregion of the downstream nucleobase polymer. As above, the presence ofthe second mobility-modifying polymer increases the sensitivity of theinvader assay by enhancing the difference between the electrophoreticmobility of the flap endonuclease generated product, i.e., the firstregion of the downstream nucleobase polymer, which comprises a label anda first mobility-modifying polymer, and the electrophoretic mobility ofthe uncleaved downstream nucleobase polymer. Accordingly, the secondmobility-modifying polymer has a molecular weight of at least 2000. Inother embodiments, the second mobility-modifying polymer has a molecularweight of at least 5,000, at least 10,000, at least 20,000, and at least100,000. In one embodiment, the second mobility-modifying polymer is amobility-modifying polymer of the present invention, while in otherembodiments, the second mobility-modifying polymer is amobility-modifying polymer of the art, which is, in one illustrative,non-limiting example, an uncharged mono methyl polyethyleneglycolpolymer.

In another aspect of the present invention, the mobility-modifiedsequence-specific nucleobase polymer serves as a cleavage substrate indetection reactions involving multiple sequential cleavage reactions, asdescribed in Hall, J. G. et al., U.S. Pat. No. 5,994,069. In thisembodiment, a first cleavage structure is formed as set forth above,except that in the present embodiment, the first nucleobase polymer isoptionally a mobility-modified sequence-specific nucleobase polymer. Thereaction mixture further includes a second target nucleic acid and athird nucleobase polymer, which is a mobility-modified sequence-specificnucleobase polymer, and further comprises at least one attached reportermolecule. The second target polynucleotide has a first, a second and athird region, wherein the first region is downstream of the secondregion, and the second region is downstream of the third region. Thethird nucleobase polymer has a 5′ portion fully complementary to thesecond region of the second target polynucleotide and a 3′ portion fullycomplementary to the third region of the second target polynucleotide.Treatment of the first cleavage structure results in release of a fourthnucleobase polymer, which has a 5′ portion complementary to the firstregion of the second target polynucleotide and a 3′ portion fullycomplementary to the second region of the second target polynucleotide.This released fourth nucleobase polymer forms a cleavage structure withthe second target polynucleotide and the third nucleobase polymer underconditions where the 3′ portion of the third nucleobase polymer and the5′ portion of the fourth nucleobase polymer remains annealed to thesecond target polynucleotide. Cleavage of the third nucleobase polymerwith a cleavage reagent generates a fifth and sixth nucleobase polymer,either or both of which comprise a reporter molecule and amobility-modifying polymer, thereby providing a digestion product havinga distinctive ratio of charge to translational frictional drag. Thefifth nucleobase polymer is released upon cleavage, while the sixthnucleobase polymer remains hybridized to the second targetpolynucleotide until dissociated by denaturation. Subsequent separationand detection of the fifth or sixth nucleobase polymer providesinformation about the presence of the first and second selectednucleotide sequence within the target nucleic acid.

In a further aspect of the present invention relating to a nucleotidesequence detection method involving multiple sequential cleavagereactions, a first cleavage structure is formed by first and secondnucleobase polymer and a selected nucleotide sequence within a targetnucleic acid, as set forth above. This aspect of the method furthercomprises a mobility-modified sequence-specific second target nucleobasepolymer, which has a first, a second, and a third region, wherein thefirst region is downstream of the second region, and wherein the thirdregion upstream of the second region, is fully self complementary andalso complementary to the second region, such that it forms a hairpinstructure under selected conditions. Cleavage of the first cleavagestructure with a cleaving reagent generates a fourth nucleobase polymer,which has a 5′-portion complementary to the first region and a3′-portion fully complementary to the second region of the nucleobasepolymer. Hybridization of the released fourth nucleobase polymer to thefirst and second regions of the mobility-modified sequence-specificnucleobase polymer forms a second cleavage structure with a displacedthird region that is complementary to the second region. Cleavage ofthis second cleavage structure generates a fifth and sixth nucleobasepolymers, either of which comprises a mobility-modifying polymer and alabel, thereby providing s digestion product having a distinctive ratioof charge to translational frictional drag, and whose separation anddetection provides information about the presence of the first targetnucleic acid and the second nucleobase polymer.

Methods for labeling and detecting the cleaved nucleobase polymers, asset forth infra., are equally applicable to the labeling and detectionof products of the cleavage reactions. Moreover, labeling of releasedcleavage products is also accomplished by extension of the product bytemplate independent polymerases, including but not limited to terminaltransferase and polyA polymerase as described in U.S. Pat. No.6,090,606, which is hereby specifically incorporated by reference.

In yet another aspect, the mobility-modified sequence-specificnucleobase polymers of the present invention are employed within ageneral method to effect the electrophoretic separation of targetnucleic acids of different sizes in non-sieving media. Normally, nucleicacids of different length, i.e. consisting of different numbers ofnucleobase residues, nevertheless display an essentially invariant ratioof charge to translational frictional drag. Accordingly, such moleculescannot be separated electrophoretically in non-sieving media. However,attachment of a mobility-modified sequence-specific nucleobase polymerof the present invention to target nucleic acids of different lengthalters their ratio of charge to translational frictional drag of thetarget nucleic acids in a manner and to a degree sufficient to effecttheir electrophoretic separation in non-sieving media. Furthermore, andin contrast to electrophoretic separations in sieving media, longernucleic acids to which a mobility-modified sequence-specific nucleobasepolymer of the present invention has been attached will migrate morerapidly than a shorter nucleic acid to which the same mobility-modifiedsequence-specific nucleobase polymer has been attached. Applicantsbelieve, although without wishing to be held to that belief, that suchseparations are based upon the proportionately smaller effect ofattachment of a mobility-modifying sequence-specific nucleobase polymerof defined mass and size to a longer chain nucleic acid molecule than toa shorter chain nucleic acid molecule. Consequently, the ratio of chargeto frictional translational drag will be greater for the longer chain,providing the longer chain nucleic acid with a greater velocity in anelectric field.

Attachment of a mobility-modified sequence-specific nucleobase polymersselected from the group consisting of Structural formulae (II) and (III)to a population of nucleic acids of different length can be accomplishedusing a variety of approaches, including but not limited to enzymaticligation or direct, synthetic incorporation of the mobility-modifyingsequence-specific nucleobase polymers of the present invention into thepopulation of nucleic acids of different lengths that are to beseparated.

In one aspect of this method, a mobility-modifying sequence-specificnucleobase polymer is enzymatically ligated to a population of nucleicacids of different length but having a common nucleotide sequence at the5′-end, as is seen within the products of a chain termination nucleicacid sequencing reaction or, effectively, in chemical cleavagesequencing reactions which are transparent to all sequences other thanthose comprising the labeled 5′-end of the nucleic acid substrate. Inthis embodiment a synthetic template oligonucleotide, having twodistinct sequence regions would be used as a template to align thehybridized 3′-end of a mobility-modifying sequence-specific nucleobasepolymer so that it would directly abut the hybridized 5′-end, which isgenerally phosphorylated, that is common to the population of nucleicacids to be separated, and permit the two molecules to be covalentlyjoined. Therefore the 5′-region of the synthetic templateoligonucleotide would consist of a nucleotide sequence complementary tothe common 5′-end sequence of the molecules to be separated, while the3′-region of the synthetic template would consist of sequencescomplementary to the 3′-end of the mobility-modifying sequence-specificnucleobase polymer to be joined. In another embodiment of this approach,the common 5′-end of the population of nucleic acids to be separatedcorresponds to that generated by a sequence-specific restrictionendonuclease. Therefore the synthetic template nucleic acid consists ofat least eight nucleobases, of which at least three would becomplementary to a common 5′-sequence of the population of molecules tobe separated. The design of such template nucleic acids, as well as theconditions under which the enzymatic joining of the hybridized targetnucleic acid and the mobility-modified sequence-specific nucleobasepolymer would be carried out, are well known to those of ordinary skillin the art. Accordingly, this embodiment of the invention is applicableto any population of molecules of different sizes, provided each has acommon 5′-end sequence of at least three nucleotides, in certainembodiments, at least four nucleotides, and in further embodiments, atleast eight nucleotides. Similar procedures, wherein the sequence commonto a population of molecules of different sizes occurs at the 3′-end,and consequently, the mobility-modifying sequence-specific nucleic to beattached has a phosphorylated 5′-end with the mobility-modifying polymerattached to the 3′-end, are also included within the scope of thepresent invention.

In a further embodiment, a mobility-modifying sequence-specificnucleobase polymer is synthesized so as to be complementary to anucleotide sequence within, for example, a sequencing vector, that isupstream of, i.e. toward the 5′-end of, the binding site of a sequencingprimer used in Sanger, enzymatic chain termination sequencing reaction.In this embodiment, the mobility-modified sequence-specific nucleobasepolymer is enzymatically ligated to the sequencing primer either beforeor after extension of the sequencing primer during a chain terminationsequencing reaction. In this embodiment, the mobility-modifiedsequence-specific nucleic acid is synthesized so that, once hybridizedto the template polynucleotide, its 3′-end would either directly abutthe 5′-end of the hybridized sequencing primer, or that 3′-end wouldhybridize to sequences upstream of the 5′-end of the sequencing primer.In the latter instance, the resulting gap is filled with a nucleic acidpolymerase and the extended molecule is then enzymatically ligated tothe sequencing primer.

Another embodiment of the invention is related to the separation anddetection of mobility-modified sequence-specific nucleobase polymers andpolynucleotides. Separation of oligonucleotides is effected byelectrophoresis, chromatography, or mass spectroscopy. In methodsemploying electrophoresis, the format may be thin flat chambers. Inanother embodiment, the separation is carried out by electrophoresis incapillary tubes. The advantage of capillary electrophoresis is efficientheat dissipation, which increases resolution and permits rapidseparation under high electrical fields. Moreover, the small diametersof the capillary tubes allow separation of numerous samples in arrays ofcapillaries.

Sieving or nonsieving media are applicable to separation ofmobility-modified nucleobase polymers including but not limited to thereaction products generated in the detection methods disclosed herein.Sieving media include covalently crosslinked matrices, such aspolyacrylamide crosslinked with bis-acrylamide (see e.g. Cohen, A. S. etal. (1988) Rapid separation and purification of oligonucleotides by highperformance capillary gel electrophoresis, Proc. Natl Acad. Sci USA 85:9660; Swerdlow, H. et al., (1990), Capillary gel electrophoresis forrapid, high resolution DNA sequencing, Nuc. Acids Res. 18 (6):1415-1419) or linear polymers, for example hydroxypropylmethylcellulose,methyl cellulose, or hydroxylethylcellulose (Zhu et al. (1992), J.Chromatogr. 480: 311-319; Nathakarnkitkool, S., et al. (1992),Electrophoresis 13: 18-31).

In one embodiment, the electrophoretic medium is a non-sieving medium.Although polynucleotides are not readily separable in a non-sievingmedium, mobility-modified nucleobase polymers and polynucleotides havedistinctive ratios of charge to translational frictional drag thatpermit separation in a non-sieving media, even when the nucleobasepolymer and polynucleotides are of the same length.

4.7 Kits

Kits of the invention comprise one or more mobility-modifiedsequence-specific nucleobase polymers. The kits may also comprise asecond nucleobase polymer, typically an oligonucleotide, which isoptionally mobility-modified, where the intended assay requires a secondoligonucleotide; for example, kits for oligonucleotide ligation assaysand PCR analysis. Similarly, kits designed for ligase chain reactionamplification will further comprise at least two additional nucleobasepolymers, which together are complementary to a diagnostic ligasereaction product. The kits further may also comprise treating reagentssuch as restriction enzymes, DNA polymerases, RNases, mismatch bindingproteins, ligases, and exonucleases. Primer extension kits appropriatefor sequencing or oligonucleotide extension assays for detecting singlenucleotide polymorphisms, may further comprise nucleoside triphosphatesand/or chain terminating nucleotides. The kit may also comprise reactionbuffers for carrying out hybridizations and enzymatic treatments.

The invention further comprises kits comprising one or more of themobility-modifying phosphoramidite reagents of present invention. One ormore of the mobility-modifying phosphoramidite reagents, in such kits,may farther comprise one or more protecting groups, reporter molecules,or ligands. Such kits may also comprise one or more solvents, reagents,or solid surface-bound nucleobase materials for use in the synthesis ofmobility-modified sequence specific nucleobase polymers.

5. EXAMPLES

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

Example 1 Synthesis of DMT-Protected Poly(ethylene oxide) AlkylPhosphoramidite

Bis(diisopropylamino)chlorophosphine was synthesized by reactingphosphorous trichloride and diisopropylamine in toluene. Fifty ml of theresulting bis(diisopropylamino)phosphine was placed in a 250 ml flaskand 3.01 ml of ethanol added slowly over a two minute period and thereaction allowed to proceed for several days. After filtering themixture and removing the solvent, the structure of thebis(diisopropylamino)ethylphosphite ester reagent was analyzed by ³¹Pand ¹H NMR (³¹P NMR in CD₃CN 125.9 ppm).

Mono dimethoxytrityl (DMT) protected pentaethylene oxide (3.0 gm, 5.5mmole) tetrazole diisopropylamine salt (0.095 gm, 0.055 mmole) weredissolved in 10 ml of methylene chloride and reacted withbis(diisopropylamino)ethylphosphite ester (1.75 gm, 7.2 mmole) for 15hours. The phosphoramidite product, DMT-pentaethyleneoxideethyl-N,N-diisopropylphosphoramidite, was washed two times withsaturated NaHCO₃ solution, followed by a third wash with saturated NaClsolution and dried over Na₂SO₄. The solution was basified by addition oftriethylamine (TEA) (100 μl), and the solvent was removed. Recoveredcrude (4.2 gm) was purified using silica gel chromatography, yielding2.8 gm product (³¹P NMR in CD₃CN 145.3 ppm). To synthesizemobility-modifying phosphoramidite reagents lacking the ethyl ester, DMTprotected tetraethylene oxide was reacted with β-cyanoethyl chloroN,N-diisopropylaminophosphite under standard conditions (see e.g.Levenson. C., et al., U.S. Pat. No. 4,914,210).

Example 2 Synthesis of Mobility-Modified Sequence-Specific NucleobasePolymers

Sequence-specific nucleobase polymers labeled at the 3′-nucleoside withtetramethyl rhodamine were synthesized on a Applied Biosystems 394synthesizer using standard phosphoramidite chemistry. To synthesizemobility-modifying nucleobase polymers, the phosphoramidite reagents ofExample 1 were reacted with the 5′ OH end of the immobilized nucleobasepolymer. Subsequent oxidation with iodine and deprotection with baseconverts β-cyanoethylphosphite linkages to phosphate diester linkageswhile ethyl phosphite linkages are converted to ethyl phosphate triesterlinkages. After cleavage from the solid support and deprotection withbase at 55° C. for 4 hrs, the nucleobase polymers were purified by highperformance liquid chromatography (HPLC). Treatment with 100 μl of 80%acetic acid for 15 min removed the final DMT protecting group. Themobility-modified nucleobase polymers were purified on a PD 10 column.

Example 3 Separation Characteristics of Mobility-ModifiedSequence-Specific Nucleobase Polymers

A series of twelve-residue nucleobase polymers (“12 mers”) weresynthesized to which were attached different mobility-modifyingmonomeric units of pentaethylene oxide via either charged phosphatediester (PEO) or uncharged ethyl phosphate triester (PEE) linkages,which were synthesized as set forth in Example 2. The relativeelectrophoretic mobility profile for each compound was evaluated byseparation by capillary electrophoresis in a non-sieving medium (AppliedBiosystems 310 Genetic Analyzer). Fluorescent internal size standardsprovided the reference markers for peak retention analysis usingGeneScan® software.

An unmodified 12-mer nucleobase polymer migrates with an apparent basesize of 24.7. Attachment of three units of pentaethylene oxidecovalently linked to the nucleobase polymer moiety through negativelycharged phosphate diester linkages, retards the mobility of the 12 merby 5.3 bases, while modification with only a single monomeric unit ofpentaethylene oxide attached through an uncharged ethyl phosphatetriester linkage retards the mobility by 8.3 bases. Furthermore, theattachment of additional pentaethylene oxide units linked throughuncharged ethyl phosphate triester linkages produces non-lineardecreases in mobility of the oligonucleotide.

TABLE 1 Oligonucleotide: Mobility Mobility change relative to 3′ TMRAlabeled 12 mer (base size) unmodified oligonucleotide 5′ OH 24.7 — 5′(PEO)₃ 30.0 5.3 5′ (PEE)₁ 33.0 8.3 5′ (PEE)₃ 60.0 35.3 5′ (PEE)₆ 116.091.3 TMRA: carboxytetramethylrhodamine PEO: phosphate diester linkedpentaethylene oxide PEE: ethyl phosphate triester linked pentaethyleneoxide

Example 4 Analysis of Synthetic Modified Oligonucleotide Products ofInvader Assay

The SNP-identifying procedure generally referred to as an invader assayis based upon flap endonuclease cleavage of structures formed by twooverlapping oligonucleotides that hybridize to a target nucleic acid(see e.g. Cooksey et al., 2000, Antimicrobial Agents and Chemotherapy44: 1296-1301). Such cleavage reactions release products correspondingto the 5′-terminal nucleotide or 5′-terminal oligonucleotide of thedownstream oligonucleotide. Where those cleavage products are labeledand can be separated from the uncleaved oligonucleotide, the invaderassay can be used to discriminate single base differences in, forexample, genomic or PCR-amplified genomic sequences.

In order to demonstrate the utility of the mobility-modifying polymersegments of the present invention for use, e.g., in invader assays, thecompounds of Table 2, which represent exemplary reaction products thatcould be generated within an invader assay, were synthesized usingmethods disclosed supra. Each of the compounds of Table 2 was labeled atthe 5′-end with a fluorescent dye as indicator. The relative mobility ofeach of these compounds was determined by capillary electrophoresisusing an Applied Biosystems instrument, model number 310, and dataanalysis was performed using GeneScan software, version 2.1fc4.

TABLE 2 Oligonucleotide: Mobility change relative to 5′ FAM labeledMobility (base size) unmodified oligonucleotide G 39.6 — 5′(PEO)₁-(PEE)₁ 75.5 35.9 5′ (PEE)₁ 95.1 55.5 5′ (PEO)₂-(PEE)₁ 65.5 25.95′ (PEE)₃ 250 210.4 5′ (PEE)₂ 160 120.4 5′ (PEO)₂-(PEE)₂ 104 64.4 5′(PEE)₁₀ 1300 1260 FAM: carboxyfluorescein PEO: phosphate diester linkedpentaethylene oxide PEE: ethyl phosphate triester linked pentaethyleneoxide

The data in Table 2 demonstrate the unexpectedly large effect onmobility provided by the mobility-modifying polymer segments of thepresent invention, especially in comparison to the phosphate diesterlinked pentaethylene oxide monomers of the art. The data of Table 2further demonstrate the extent of electrophoretic separation that can beobtained using the mobility-modifying phosphoramidite functionalizingreagents of the present invention. Moreover, the data of Table 2 alsodemonstrate that compounds of intermediate mobility are obtained bycombining the mobility-modifying phosphoramidite functionalizingreagents of the present invention with, as a non-limiting example,phosphate diester linked pentaethylene oxide monomers of the art.

Example 5 Analysis of Cleaved Modified Oligonucleotide Products ofInvader Assay

An invader assay probe is synthesized that comprises a fluorescent dye(FAM) coupled to a first mobility-modifying polymer, a dimer of amobility-modifying phosphoramidite functionalizing reagent of thepresent invention ((PEE)₂), linked to the following oligonucleotide:5′-GGGACGGGGTTCAGC-3′-NH₂, using standard DNA synthesis methods and PEEphosphoramidite reagents. After cleavage from the support anddeprotection with base at 55° C. for 4 hours, the oligonucleotide ispurified by HPLC. The oligonucleotide:5′-FAM-(PEE)₂-GGGACGGGGTTCAGC-3′-NH₂ is dissolved in water, coupled witha second mobility-modifying polymer, mono-methyl polyethylene glycol5000 propionic acid N-succinimidyl ester (Fluka) in the presence ofNaHCO₃ for two hours, and purified by HPLC, yielding the derivatived,mobility-modified product 5′-FAM-(PEE)₂-GGGACGGGGTTCAGC-3′-PEG 5000.

The invader assay is performed using, as template, a 527 bp PCR productgenerated by amplification of a segment of human genomic DNAcorresponding to the TNF-α gene, using the following PCR primers:5′-GAGTCTCCGGGTCAGAATGA (forward) and 5′-TCTCGGTTTCTTCTCCATCG (reverse).In the first step of the invader assay, approximately 0.2 pmole of thePCR product is denatured at 95° C. for 5 min. in the presence of 0.5pmole of invading probe (5′-GAGGCAATAGTTTTTGAGGGGCATGT). In the secondstep, 50 ng of Cleavase VII is added along with 10 pmole of5′-mobility-modified oligonucleobase probe,5′-FAM-(PEE)₂-GGGACGGGGTTCAGC-3′-PEG 5000, in a total reaction volume of10 μl further comprising 10 mM MOPS, pH 8.0, 7.5 mM MgCl₂, 0.05% Tween20, and 0.05% Nonidet P40. The invader assay is incubated for 15 hoursat 66° C. The reaction is terminated and a 1 μl aliquot thereof iselectrophoresed on an Applied Biosystems instrument, model number 310,with data analysis performed using GeneScan software, version 2.1fc4.Analysis of the results demonstrates the presence of the cleavageproduct 5′-FAM-(PEE)₂-G, which is well separated from the uncleavedprobe, 5′-FAM-(PEE)₂-GGGACGGGGTTCAGC-3′-PEG 5000. The uncleaved probewas detected as a plurality of closely-spaced peaks arising from theplurality of molecular weight species included within thecommercially-available mono methyl polyethylene glycol polymer productattached to the 3′-end of the labeled probe,5′-FAM-(PEE)₂-GGGACGGGGTTCAGC-3′ as the second mobility-modifyingpolymer.

All publications and patents referred to herein are hereby incorporatedby reference in their entirety. As recognized by those skilled in theart of molecular biology, the use of mobility-modified sequence-specificnucleobase polymers are adaptable to a variety of methods. Variousmodifications and variations of the above described method andcomposition will be apparent to those skilled in the art withoutdeparting from the spirit and scope of the invention. As a specificexample, although various embodiments of the invention may bedescriptively exemplified with DNA or RNA oligonucleotides, skilledartisans will recognize that the described embodiments may also workwith other nucleobase polymers, including analogs and derivatives of RNAand DNA oligonucleotides. Although specific preferred embodiments of theclaims are described, the invention as claimed should not be limited tothe specific embodiments. Various modification of the described modeswhich are obvious to those skilled in the art are intended to be withinthe scope of the following claims.

What is claimed is:
 1. A mobility-modified sequence-specific nucleobasepolymer comprising a mobility-modifying polymer linked to asequence-specific nucleobase polymer, according to Structural formula(II) or (III):

or a salt thereof, wherein: R² is selected from the group consisting ofalkyl comprising at least two carbon atoms, aryl, (R⁸)₃Si— where each R⁸is independently selected from the group consisting of linear andbranched chain alkyl and aryl, base-stable protecting groups, andR⁵—X—[(CH₂)_(a)—O]_(b)—(CH₂)_(a)—; each R¹⁰ is independently selectedfrom the group consisting of hydrogen and R²; R⁵ is selected from thegroup consisting of hydrogen, protecting group, reporter molecule, andligand;

each R⁴ is independently selected from the group consisting of hydrogenand R²; each X is independently selected from the group consisting of O,S, NH and NH—C(O); each a is independently an integer from 1 to 6; eachb is independently an integer from 0 to 40; each d is independently aninteger from 1 to 200; and OLIGO comprises a sequence-specificnucleobase polymer, with the proviso that at least one R¹⁰ or at leastone R⁴ is other than hydrogen, wherein the mobility-modifying polymercomprises at least one phosphotriester linkage.
 2. The mobility-modifiedsequence-specific nucleobase polymer of claim 1 in which each X is O. 3.The mobility-modified sequence-specific nucleobase polymer of claim 1 inwhich each a is
 2. 4. The mobility-modified sequence-specific nucleobasepolymer of claim 3 in which each b is
 4. 5. The mobility-modifiedsequence-specific nucleobase polymer of claim 1 in which OLIGO is a DNA,RNA, DNA analog, or RNA analog oligonucleotide.
 6. The mobility-modifiedsequence-specific nucleobase polymer of claim 1 in which OLIGO is ananalog of a DNA or RNA oligonucleotide.
 7. The mobility-modifiedsequence-specific nucleobase polymer of claim 1 in which OLIGO comprisesat least one non-negatively charged internucleotide linkage.
 8. Themobility-modified sequence-specific nucleobase polymer of claim 7,wherein said internucleotide linkage is a mono alkyl phosphate triester.9. The mobility-modified sequence-specific nucleobase polymer of claim 1in which R⁵ is a reporter molecule.
 10. The mobility-modifiedsequence-specific nucleobase polymer of claim 9 in which the reportermolecule is a fluorophore, a chemiluminescent moiety, or a ligand. 11.The mobility-modified sequence-specific nucleobase polymer of claim 1 inwhich OLIGO includes a detectable label.
 12. The mobility-modifiedsequence-specific nucleobase polymer of claim 9 in which the detectablelabel is a fluorophore.
 13. The mobility-modified sequence-specificnucleobase polymer of claim 1 in which OLIGO comprises a polyethlyeneoxide polymer.
 14. The mobility-modified sequence-specific nucleobasepolymer of claim 13, wherein the polyethlyene oxide polymer is a monomethyl polyethlyene oxide polymer.
 15. The mobility-modifiedsequence-specific nucleobase polymer of claim 13, wherein thepolyethlyene oxide polymer has a molecular weight of at least 2000daltons.
 16. The mobility-modified sequence-specific nucleobase polymerof claim 13, wherein the polyethlyene oxide polymer has a molecularweight of at least 5000 daltons.
 17. The mobility-modifiedsequence-specific nucleobase polymer of claim 1, wherein themobility-modifying polymer is attached to the 5′-end of thesequence-specific nucleobase polymer.
 18. The mobility-modifiedsequence-specific nucleobase polymer of claim 17, further comprising apolyethlyene oxide polymer attached to the 3′-end of thesequence-specific nucleobase polymer.
 19. The mobility-modifiedsequence-specific nucleobase polymer of claim 18, wherein thepolyethlyene oxide polymer is a mono methyl polyethlyene oxide polymer.20. The mobility-modified sequence-specific nucleobase polymer of claim18, wherein the polyethlyene oxide polymer has a molecular weight of atleast 2000 daltons.
 21. The mobility-modified sequence-specificnucleobase polymer of claim 18, wherein the polyethlyene oxide polymerhas a molecular weight of at least 5000 daltons.
 22. Themobility-modified sequence-specific nucleobase polymer of claim 1,wherein the mobility-modifying polymer is attached to the 3′-end of thesequence-specific nucleobase polymer.
 23. The mobility-modifiedsequence-specific nucleobase polymer of claim 22, further comprising apolyethlyene oxide polymer attached to the 5′-end of thesequence-specific nucleobase polymer.
 24. The mobility-modifiedsequence-specific nucleobase polymer of claim 22, wherein thepolyethlyene oxide polymer is a mono methyl polyethlyene oxide polymer.25. The mobility-modified sequence-specific nucleobase polymer of claim22, wherein the polyethlyene oxide polymer has a molecular weight of atleast 2000 daltons.
 26. The mobility-modified sequence-specificnucleobase polymer of claim 22, wherein the polyethlyene oxide polymerhas a molecular weight of at least 5000 daltons.
 27. A compositioncomprising a mixture of different mobility-modified sequence-specificnucleobase polymers, in accordance with claim 1, wherein each differentnucleobese polymer has a distinctive ratio of charge to translationalfrictional drag relative to the friction drags of the other differentnucleobase polymers.
 28. The composition of claim 27, wherein OLIGO ineach different mobility-modified-specific nucleobase polymer has thesame number of nucleobase units.
 29. A mobility-modifyingphosphoramidite reagent having the structure:

wherein: R² is selected from the group consisting of alkyl comprising atleast two carbon atoms, aryl, (R⁸)₃Si— where each R⁸ is independentlyselected from the group consisting of linear and branched chain alkyland aryl, base-stable protecting groups, andR⁵—X—[(CH₂)_(a)—O]_(b)—(CH₂)_(a)—; R⁵ is selected from the groupconsisting of hydrogen, protecting group, reporter molecule, and ligand;R⁶ and R⁷ are each independently selected from the group consisting ofC₁-C₆ alkyl, C₃-C₁₀ cycloalkyl, C₆-C₂₀ aryl, and C₂₀-C₂₇ arylalkyl; X isselected from the group consisting of O, S, NH, NH—C(O); each a isindependently an integer from 1 to 6; and b is an integer from 0 to 40.30. The mobility-modified sequence-specific nucleobase polymer of claim1 wherein R² is chosen from ethyl, n-propyl, isopropyl, n-butyl,tert-butyl, and neopentyl.
 31. The mobility-modified sequence-specificnucleobase polymer of claim 1 wherein R² is ethyl.
 32. Themobility-modified sequence-specific nucleobase polymer of claim 1wherein R² is n-propyl.
 33. The mobility-modified sequence-specificnucleobase polymer of claim 1 wherein R² is isopropyl.
 34. Themobility-modified sequence-specific nucleobase polymer of claim 1wherein R² is n-butyl.
 35. The mobility-modified sequence-specificnucleobase polymer of claim 1 wherein R² is tert-butyl.
 36. Themobility-modified sequence-specific nucleobase polymer of claim 1wherein R² is neopentyl.
 37. The mobility-modified sequence-specificnucleobase polymer of claim 1 wherein each R¹⁰ is independently chosenfrom hydrogen, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, andneopentyl.
 38. The mobility-modified sequence-specific nucleobasepolymer of claim 1 wherein each R¹⁰ is independently chosen fromhydrogen and ethyl.
 39. The mobility-modified sequence-specificnucleobase polymer of claim 1 wherein each R¹⁰ is independently chosenfrom hydrogen and n-propyl.
 40. The mobility-modified sequence-specificnucleobase polymer of claim 1 wherein each R¹⁰ is independently chosenfrom hydrogen and isopropyl.
 41. The mobility-modified sequence-specificnucleobase polymer of claim 1 wherein each R¹⁰ is independently chosenfrom hydrogen and n-butyl.
 42. The mobility-modified sequence-specificnucleobase polymer of claim 1 wherein each R¹⁰ is independently chosenfrom hydrogen and tert-butyl.
 43. The mobility-modifiedsequence-specific nucleobase polymer of claim 1 wherein R¹⁰ isindependently chosen from hydrogen and neopentyl.
 44. Themobility-modified sequence-specific nucleobase polymer of claim 1wherein each R⁴ is independently chosen from hydrogen, ethyl, n-propyl,isopropyl, n-butyl, tert-butyl, and neopentyl.
 45. The mobility-modifiedsequence-specific nucleobase polymer of claim 1 wherein each R⁴ isindependently chosen from hydrogen and ethyl.
 46. The mobility-modifiedsequence-specific nucleobase polymer of claim 1 wherein each R⁴ isindependently chosen from hydrogen and n-propyl.
 47. Themobility-modified sequence-specific nucleobase polymer of claim 1wherein each R⁴ is independently chosen from hydrogen and isopropyl. 48.The mobility-modified sequence-specific nucleobase polymer of claim 1wherein each R⁴ is independently chosen from hydrogen and n-butyl. 49.The mobility-modified sequence-specific nucleobase polymer of claim 1wherein each R⁴ is independently chosen from hydrogen and tert-butyl.50. The mobility-modified sequence-specific nucleobase polymer of claim1 wherein each R⁴ is independently chosen from hydrogen end neopentyl.51. The mobility-modified sequence-specific nucleobase polymer of claim29 wherein R² is chosen from ethyl, n-propyl, isopropyl, n-butyl,tert-butyl and neopentyl.
 52. The mobility-modified sequence-specificnucleobase polymer of claim 29 wherein R² is ethyl.
 53. Themobility-modified sequence-specific nucleobase polymer of claim 29wherein R² is n-propyl.
 54. The mobility-modified sequence-specificnucleobase polymer of claim 29 wherein R² is isopropyl.
 55. Themobility-modified sequence-specific nucleobase polymer of claim 29wherein R² is n-butyl.
 56. The mobility-modified sequence-specificnucleobase polymer of claim 29 wherein R² is tert-butyl.
 57. Themobility-modified sequence-specific nucleobase polymer of claim 29wherein R² is neopentyl.